JP6947533B2 - Use of AT (n) inserts in promoter elements to control the expression level of coding sequences in plants - Google Patents

Description

The present invention is at a particular point in a promoter region sequence to regulate the expression level of a coding sequence in a plant that can be artificially upregulated (upstream) or downregulated (downstream) within certain restrictions. AT _(n) Regarding the use of inserts.

The promoter region of a gene is generally contained in a DNA sequence having 1 to 100 and 200 base pairs upstream from the gene transcription initiation site, typically containing one or more transcription factor binding sites and / or it. Adjacent. All genes have transcriptional regulation upstream (upper) and downstream (lower) of the transcription initiation site. Transcription factors recognize and bind to these regulatory sequences and control the synthesis of transcribed messenger RNA. These sequences include promoters, enhancers and regulatory sequences. Promoters are used in gene constructs and, in a constitutive manner, can be engineered by genetic engineering means and induced by certain environments in order to overexpress, inhibit, or regulate their expression.

Various techniques for overexpressing genes are currently available in the field of biotechnology, of which the main technique is the constitutive promoter 35S isolated from cauliflower mosaic virus (CaMV). Contains constructs of expression cassettes. However, there are some exceptions to the use of this promoter in gene constructs. Presence of potential risk of genetic recombination between viral genomes infecting plants, or more precisely, between messenger RNA transcripts, due to the use of short-stranded viral DNA and its inserts into the plant genome. Was clarified experimentally. This risk leads to the creation of new viral strains, preferably more malignant types.

Promoter 35S CaMV is thought to be accidental and functions effectively in all plants and in green algae, yeast and E. coli. It has a modular structure with interchangeable pairs in common with other plant and animal virus promoters. It also has recombinant hotspots, which are positioned by multiple triggers involved in the recombination process similar to other recombinant hotspots and are often used in transgenic plants of the Agrobacterium species. Boundaries of T-DNA vectors are included. This hotspot tends to divide and bind to other DNA, thereby increasing the potential for horizontal gene transfer and recombination processes. Therefore, insertion of invasive exogenous DNA into the genome, reactivation of dormant viruses and novel viruses that are only stable if they contribute some selective advantages compared to wild-type viruses. Due to development, potential risks are mutagenesis and carcinogenesis.

Partial or complete transcriptional events and / or inhibition of endogenous homologous expression were observed using the promoter 35S CaMV (Napoli et al (1990) The Plant Cell 2: 279-289; Van der Kroll et al (1990). ) Plant Cell 2 291-299). This inactivation occurs due to a complex mechanism involving methylation and epigenetic changes (Renckens et al (1992) Mol. Gen. Genet. 233: 53-64; Neuhuber et al (1994) Mol. Gen. Genet. 244: 230-241; Meyer & Heidmann (1994) Mol. Gen. Genet. 243: 390-399; Thierry & Vaucheret (1996) Plant Mol. Biol. 32: 1075-1083). The actual mechanism of directing certain sequences to methyltransferases or other enzymes involved in epigenetic alterations remains unknown, but the mutual dependence of DNA-DNA, DNA-RNA and RNA-RNA on each other. There is a hypothesis that it suggests the involvement of action (Grierson et al. (1991) Trends in Biotechnology 9: 122-123); Mattzke & Mattzke (1995) Plant Physiologic 107: 679-685).

In the literature, there are data demonstrating that the CaMV gene was integrated into the canola chromosome recombinantly in the genome of the infectious virus, resulting in multiple novel malignant viruses. These recombinations between CaMV viruses include promoter regions (Vaden & Melcher (1992) Virology 177: 111) and can occur between DNA and DNA, or between RNA and RNA, often in the parental. It causes infections that are more severe than their counterparts (Mol. Plant-Microbe Interactions 5:48, 1992).

In addition, the use of promoter 35S CaMV usually results in expression of the product of the endogenous gene at a ratio of less than 1% of the total protein. Some improvements in this promoter, such as duplication of a sequence or the addition of enhancers, increased its expression, but for some applications the expression level of the endogenous gene product needs to be further increased. ..

In general, with respect to the characteristics of resistance to biological and abiotic stress, in experiments performed with the constitutive promoter 35S CaMV to obtain genetically engineered plants, this resistance increased in some cases. However, one of the undesired effects derived from the use of this promoter is a reduction in plant size, which usually affects plant growth and development and affects small plants regardless of the transformed plant species. To reach (Kasuga et al. (1999) Nature America Inc. 287-291).

Therefore, certain negative effects, such as delays in plant growth, may occur, but strategies such as the use of stress-induced promoters that regulate gene expression may prevent or mitigate such effects. be.

In another task, Gelvin et al. (Plant Molecular Biology Manual. Norwell, MA: Kruwer Acidemic Publicers, 1995), Bacterial Agrobacterium tumefaciens (Agrobacterium tumefaciens (Agrobacterium tumefaciens) (Agrobacterium tumefaciens) The expression of GUS detected by several promoters constructed using various combinations that regulate the sequence of synthase (mas) was analyzed. Among the various combinations, the hybrid promoter formed by three iterations of the ocs gene activation sequence fused using the mas activation element and the mas promoter region is called "(Aocs) 3AmasPmas" and is called the promoter CaMV. The GUS marker gene, which is 10-fold larger than cells containing 35S, was presented at the expression level of transformed tobacco cells. This novel promoter did not affect transformation efficiency, but high levels of marker gene expression facilitated the identification of larger proportions of transformed cells. Expression levels were high in leaves, roots, and various cell types. In addition, this promoter was very active in manioc and pea plants, and it was difficult to transform the two grains with Agrobacterium species. This group further described the success in the study of rapid transcription of genes introduced into tobacco and corn plants (after 18 hours) using this promoter "(Aocs) 3AmasPmas" and regulated expression by the promoter CaMV 35S. Unlike, its activity is weak and does not allow detection of any gene expression within this period.

There is an additional option to use a stress-inducible promoter for gene overexpression in the gene construct, in which respect the promoter rd29 isolated from Arabdopsis thaliana is good for abiotic stress. Most used to search for resistant plants. In general, these gene constructs are usually fused to family DREB (dehydration response element binding) genes that regulate gene expression in response to drought, salt, and environmental stresses such as low and high temperatures. In some tasks with different plant species, plants resistant to abiotic stress were reported as a result of genetic transformation with induced stress promoters (Oono et al. (2003) The Plant Journal). , 34: 868-887; Kasuga et al. (2004) Plant Cell Physiol 45 (3): 346-350).

Fuganti et al. (2004) used a soybean genotype that is resistant and susceptible to cyst nematodes. This document contains initial data on molecular markers used in the supportive selection of soybean genotypes resistant to cyst nematodes. However, no type of molecular difference between the tested individuals was detected with respect to the level of the DNA sequence or the expression of the mRNA encoded by this gene.

Fuganti (Master's Thesis, State University of Londrina [University University of Londrina], 2004) shows the selection of resistant strains, here the resistance genes of the genomic region including the gene of interest The molecular marker used was used. This study does not disclose qualitative and quantitative data showing possible correlations between the size _{of AT (n)} inserts in gene promoters and the obtained levels of expression of their mRNA or protein. The present invention proposes the ability to regulate and / or upregulate or downregulate any expression level, provided that a certain number of AT _{(n) inserts have been inserted into the promoter region.} No such proposal has been presented and tested in this study.

Fuganti (PhD Qualitative, State University of Maringa [University de Maringa], 2007) is a variety of _{AT (n)} inserts of different sizes within the promoter region of the gene Gmhsp17.6-L, as well as different sized AT (n) inserts within the promoter region of the gene Gmhsp17.6-L. Quantitative and qualitative data showing possible correlations with their expression levels in response to target stress (cold, heat, drought, salt, infection by nematode larvae) are presented. _{However, the coding region may be fused to promoter regions containing different AT (n)} sizes in the absence and / or presence of biological and abiotic stress, upregulating or upregulating the expression level of any gene. The possibility of downward control has not been shown and considered.

US Pat. Provide a method of using heat generation. The proposed invention regulates the expression level of any gene fused to the promoter region of a heat shock protein with _{AT (n)} inserts of various sizes expressed in all transformed cells in the gene sequence. And the purpose is to control upwards or downwards. Biological or abiotic stress may be applied to the entire plant. Any indication of the role of AT inserts in the regulation of gene expression and their availability as a tool for controlling arbitrary gene expression, provided that the same is inserted into its promoter region, is disclosed. Not.

Japanese Patent Application Laid-Open No. 2003190706 relates to an endogenous gene fused to a DNA sequence exhibiting promoter activity that promotes an increase in the total amount of a gene product (protein). This region with promoter activity was generated from mutation alterations, including substitutions and deletions of nitrogen-conjugated bases. The present invention requires the use of a complete gene promoter, including all the genetic elements required to induce mRNA synthesis. It is clear in the present invention that regulation of gene expression results in levels differentiated according to the size of the _{AT (n)} insert, rather than random mutations in the promoter region of the gene.

Patent Document Europe No. 1930423 discloses promoter regions of heat shock proteins divided into different final sizes used in systems for producing heterologous proteins whose expression levels are not regulated by other conventional promoters. Specific abiotic or chemical stresses are used to activate expression. The present invention uses an existing variation in the genotype tested, a fragment smaller than 1000 bp used in reference EP1930423, which is detected in the promoter region of the gene Gmhsp17.6-L. This variation is provided by a different number of AT (n) inserts in a particular region of the promoter, resulting in a size variation between _{312 bp (AT 9} ) and 358 bp (AT _{33) depending on the AT (n) insert.}

Patent Document US No. 5929302 uses a transient tissue-specific promoter that regulates gene expression during maturation in tissues such as leaves and tentacles. The promoter was isolated from the dru gene itself in raspberries or from its homologues to express the chimeric gene. This promoter and specific genes, such as genes that control color and scent changes, genes for enzymes or catalytic products that modify plant cell processes, genes whose products affect ethylene synthesis, alternative fungal regulatory genes , And suggests a combination with the sucrose accumulation gene. The present invention has changes in the promoter region of any gene in order to change the expression of any gene in all cells and throughout the life cycle of the plant. Variations in the expression level of the gene product _{correlate with different numbers of AT (n)} inserts into the promoter and, as a result, with promoters of different sizes. The present invention proposes to regulate arbitrary gene expression, including these alterations.

Therefore, the present invention is innovative because it differs from all currently existing methods available in scientific papers that overexpress genes. To do so, the present invention can use the insertion of AT sequences with many and few repetitions to allow regulation of the level of gene expression and increase or decrease it.

The present invention relates to the use _{of AT (n)} inserts in promoter elements to regulate the expression level of coding sequences in plants. The present invention also relates to a gene expression cassette containing a promoter region of a gene with a different number of AT inserts fused to a GUS protein to transform a soybean embryo.

The present invention provides a method of controlling the expression level of a coding sequence in a plant.
(I) Stable transformation of plant cells using an expression cassette carrying an _{AT (n)} insert in a promoter element operably linked to a gene of interest;
(Ii) Stable transformed plant cells are cultured under plant cell proliferation conditions;
(Iii) The cassette of (i) comprises regenerating a transgenic plant that is stably integrated into its genome, wherein the transgenic plant exhibits different levels of gene expression when compared to a control plant. show.

In addition, the present invention provides an expression cassette containing two promoter regions with different numbers of AT inserts linked to the promoter region of the Gmhsp17.6-L gene.

In another aspect of the invention, a method for obtaining a genetically modified plant containing _{an AT (n) -insert is provided, wherein the method is:}
(I) Stable transformation of plant cells using an expression cassette carrying an _{AT (n)} insert within a promoter element operably linked to a gene of interest;
(Ii) Stable transformed plant cells are cultured under plant cell proliferation conditions;
(Iii) The method comprising regenerating a stably genetically modified plant.

The result is that the expression level of certain genes is artificially upregulated (upstream) within certain limits by inserting _{AT (n) repeated bases into the promoter region via genetic engineering tools.} ) Or downward control (downstream).

Agarose gels using amplification of the microsatellite marker SOYHSP176 are shown in the resistant parent sample PI595099, the sensitive parent sample BRS133, and the individuals of the resistant population (Sample 01-Sample 05) and the sensitive sample (Sample 06-Sample 10). Arrows indicate bunts caused by marker amplification in susceptible parents and are also found in susceptible populations. The sequence of the gene Gmhsp17.6-L available from GenBank (accession number: M11317) is shown. Underline, primer pSoyHspPstI F and pSoyHsp-BamHI R defines an amplified fragment of the promoter region. The dot box is the AT _(n) insert, and the gray box is the start codon (ATG) and stop codon (TAA) of the coding sequence. The sequence of the gene Gmhsp17.6-L (GenBank accession number: M11317) is shown. The gray boxes highlight the start codons (ATG) and stop codons (TAA) that define the coding sequence, the point boxes highlight the AT (n) inserts in the promoter region, and the underline is designed for complete gene sequencing. Primer pSoyHspPstI F and SoyHSP Cl It is R. Primer pSoyHsp designed based on the complete sequence of genes available from GenBank (access number: M11317) AlleI F and pSoyHsp NcoI The amplification of the promoter region of the gene Gmhsp17.6-L using R is shown. The ladders are 100pb and 500pb. Amplification of the complete gene Gmhsp17.6-L is shown using primers pSoyHSP PstIF and pSoyHSP Cl R designed based on the complete sequence of the gene available from GenBank (access number: M11317). The ladders are 100 bp and 500 bp. Primer pSoyHSPPstI that defines the promoter region F and pSoyHSPBamHI Gene Gmhsp17.6-L generated by amplification of resistant parent PI595099, susceptible parent BRS133, susceptible population 256-S, 259-S, and resistant populations JF7002, JF7027, and JF7056 using the set of R. The alignment of the sequence of the promoter region is shown. Primers were defined based on the complete sequence of the gene Gmhsp17.6-L (GenBank accession number: M11317). The point box is an AT _(n) insert. The box is probably a TATA-box. The black box with white letters is one of the heat shock elements (HSE) of the consensus sequence 5'AGGAAnnTTCT3'. In the gray box with white letters, there is another consensus sequence 5'cTTCtaGAAgcTTCtaGAAg3' with core 5'CTnGAAnnTTCnAG3'. The gene sequence present between the promoter region of gene Gmhsp17.6-L and the initiation of the coding sequence compared to the sequence of the gene available in GenBank (acceptance number: M11317) is shown, and the material of the study is susceptibility (BRS133). ) And resistance (PI595099), individuals of the susceptible populations 256-S, 259-S and 266-S, and individuals of the resistant populations JF7002, JF7027 and JF7056. Nucleotides in the gray box highlight the codon ATG at the transcription initiation site of the coding region. Sensitive parent BRS133 and resistant PI595099, susceptible populations 256-S, 259-S and 266-S individuals, and resistant for all materials under study compared to the sequence of the gene available in GenBank (accession number: M11317). The alignment of the coding sequence of the gene Gmhsp17.6-L derived from the individuals of the populations JF7002, JF7027 and JF7056 is shown. Nucleotides in the gray box highlight the ATG start codon and TAA thermal transcription codon that define the coding region. Sensitive parent BRS133 and resistant PI595099, susceptible populations 256-S, 259-S and 266-S individuals, and resistant for all materials under study compared to the sequence of the gene available in GenBank (accession number: M11317). The alignment of the coding sequence of the gene Gmhsp17.6-L derived from the individuals of the populations JF7002, JF7027 and JF7056 is shown. Nucleotides in the gray box highlight the ATG start codon and TAA thermal transcription codon that define the coding region. Sensitive parent BRS133 and resistant PI595099, susceptible populations 256-S, 259-S and 266-S individuals, and resistant for all materials under study compared to the sequence of the gene available in GenBank (acceptance number: M11317). The alignment of the amino acid sequence encoded by the coding sequence of the gene Gmhsp17.6-L derived from the individuals of the populations JF7002, JF7027 and JF7056 is shown. M. Ribonuclease protection performed with a probe (326 pb) constructed from a fragment of the gene Gmhsp17.6-L (GenBank accession number: M11317) using Javanica resistant and sensitive samples is shown. The number of upper parts is equivalent to the sample used: susceptible parent (BRS133) and resistant (PI595099) and M. et al. Inoculated with Javanica eggs (2 and 4), kit positive controls (indicated by 5-arrows), RNase enzyme controls, designed probes (6), and RNase undigested (7). be. Cyst nematode M. Relative quantification of the gene Gmhsp17.6-L in resistant (PI595099) and susceptibility (BRS133) parents in Javanica inoculated and non-inoculated treatments is shown. Data were obtained by real-time PCR with primers SOYHSP PSC F and SoyHSP PSC R designed using the program Primer Express® software v20 using the complete sequence of the gene available in GenBank (acceptance number: M11317). .. Quantification was performed using the 2- ^{ΔΔCt method.} The gene rRNA 18S was used as a gauge and as a non-inoculated sample. Samples were amplified in three-point measurements in three independent runs using the SYBR method. Cyst nematode M. Relative quantification of the gene Gmhsp17.6-L in individuals of susceptible populations 256-S, 259-S and 266-S; and resistant populations JF7002, JF7027 and JF7056 in inoculated and non-inoculated treatment of Javanica (x) Is shown. Data were obtained by real-time PCR with primers SOYHSP PSC F and SoyHSP PSC R designed using the program Primer Express® software v20 using the complete sequence of the gene available in GenBank (acceptance number: M11317). .. Quantification was performed using the 2- ^{ΔΔCt method.} The gene rRNA 18S was used as a gauge and as a non-inoculated sample 256-S. Samples were amplified in three-point measurements in three independent runs using the SYBR method. A schematic model of plasmid pAG1 illustrating the Gus reporter gene encoding the enzyme β-glucuronidase, the gene Ahas and the promoter act2 is shown. It also shows a limit map. Materials tested: In the promoter region of the gene Gmhsp17.6-L of all constructs of the expression cassette obtained using BRS133, PI595099, 256-S, 259-S and 266-S, JF7002, JF7027 and JF7056. Amplification via PCR is shown. The reaction is a primer pSoyHspPSTI that amplifies a fragment of 363 pb. F and pSoyHSPBamHI It was performed using R and then separated by electrophoresis on an agarose gel (1.0%). The amplification reaction between sample BRS133 and JF7056 was concentrated during PCR. Materials tested: Part of the gene Ahas present in plasmid pAG1 of all constructs of expression cassettes obtained using BRS133, PI595099, 256-S, 259-S and 266-S, JF7002, JF7027 and JF7056. Amplification via PCR is shown. The reaction is a primer Ahas1 that amplifies a fragment of 654 pb. F and Ahas2 It was performed using R and then separated by electrophoresis on an agarose gel (1.0%). Materials tested: PCR of terminal region Nos present in plasmid pAG1 of all constructs of expression cassettes obtained using BRS133, PI595099, 256-S, 259-S and 266-S, JF7002, JF7027 and JF7056. Shows amplification via. The reaction is primer Nos1 which amplifies a fragment of 200 pb. F and Nos3 It was performed using R and then separated by electrophoresis on an agarose gel (1.0%). Shows a schematic model exemplifying the structure of abiotic and biological stress test plaques, to which they are sensitive to cyst nematodes, BRS133 and Pintado (spots) transformed with different expression cassettes. The varieties of soybean embryos were fed. CN-negative control, untransformed embryo; CP-positive control, plasmid pAG1-transformed embryo; embryo transformed with an expression cassette constructed using a promoter region of S-sensitive parental origin , PAG1 / promoter GmHSP BRS133; embryos transformed with an expression cassette constructed using promoter regions of individual origin in the R-resistant population, pAG2 / promoter region GmHSP JF7027. Sensitive to cyst nematode Javanica, expression cassette pAG1 / promoter GmHsp BRS133 and pAG1 / promoter Gmhsp Tissues of soybean varieties BRS133 and pintado embryos transformed with biovaritics using JF7027 and subjected to heat stress at temperatures of 25 ° C. for 2 hours, 4 hours and 24 hours. The chemical test is shown. Blue dots indicate a positive reaction. CN-negative control, untransformed embryo; CP-positive control, embryo transformed with plasmid pAG1; S-expression cassette pAG1 / promoter Gmhsp Sensitive embryos transformed with BRS133; R-expression cassette pAG1 / promoter Gmhsp Resistant embryos transformed with JF7027. Sensitive to cyst nematode Javanica, expression cassette pAG1 / promoter GmHsp BRS133 and pAG1 / promoter Gmhsp Histochemical studies performed on embryos of the soybean variety BRS133 and pintado (spots) transformed with biovaritics using JF7027 and subjected to heat stress at temperatures of 35 ° C. for 2 hours, 4 hours and 24 hours. Is shown. Blue dots indicate a positive reaction. CN-negative control, untransformed embryo; CP-positive control, embryo transformed with plasmid pAG1; S-expression cassette pAG1 / promoter Gmhsp Sensitive embryos transformed with BRS133; R-expression cassette pAG1 / promoter Gmhsp Resistant embryos transformed with JF7027. Sensitive to cyst nematode Javanica, expression cassette pAG1 / promoter GmHsp BRS133 and pAG1 / promoter Gmhsp Histochemical tests performed on embryos of the soybean variety BRS133 and pintado (spots) transformed with biovaritics using JF7027 and subjected to heat stress at temperatures of 45 ° C. for 2 hours, 4 hours and 24 hours. Is shown. Blue dots indicate a positive reaction. CN-negative control, untransformed embryo; CP-positive control, embryo transformed with plasmid pAG1; S-expression cassette pAG1 / promoter Gmhsp Sensitive embryos transformed with BRS133; R-expression cassette pAG1 / promoter Gmhsp Resistant embryos transformed with JF7027. Sensitive to cyst nematode Javanica, expression cassette pAG1 / promoter Gmhsp BRS133 and pAG1 / promoter Gmhsp Histochemical tests performed on embryos of soybean variety BRS133 and pintado (spots) transformed by biovaritics using JF7027 and subjected to heat stress at temperatures of 2 hours, 4 hours and 24 hours at 4 ° C. Is shown. Blue dots indicate a positive reaction. CN-negative control, untransformed embryo; CP-positive control, embryo transformed with plasmid pAG1; S-expression cassette pAG1 / promoter Gmhsp Sensitive embryos transformed with BRS133; R-expression cassette pAG1 / promoter Gmhsp Resistant embryos transformed with JF7027. Sensitive to cyst nematode Javanica, expression cassette pAG1 / promoter Gmhsp BRS133 and pAG1 / promoter Gmhsp Histochemical tests performed on embryos of the soybean variety BRS133 and pintado (spots) transformed with biovaritics using JF7027 and subjected to heat stress at temperatures of 15 ° C. for 2 hours, 4 hours and 24 hours. Is shown. Blue dots indicate a positive reaction. CN-negative control, untransformed embryo; CP-positive control, embryo transformed with plasmid pAG1; S-expression cassette pAG1 / promoter Gmhsp Sensitive embryos transformed with BRS133; R-expression cassette pAG1 / promoter Gmhsp Resistant embryos transformed with JF7027. Sensitive to cyst nematode Javanica, expression cassette pAG1 / promoter GmHsp BRS133 and pAG1 / promoter Gmhsp The histochemical tests performed on embryos of the soybean cultivar BRS133 and pintado (spots) transformed with JF7027 by biovaritics and subjected to a salt _{of NaCl 2} at 200 mM and 400 mM concentrations for 24 hours are shown. Blue dots indicate a positive reaction. CN-negative control, untransformed embryo; CP-positive control, embryo transformed with plasmid pAG1; S-expression cassette pAG1 / promoter Gmhsp Sensitive embryos transformed with BRS133; R-expression cassette pAG1 / promoter Gmhsp Resistant embryos transformed with JF7027. Sensitive to cyst nematode Javanica, expression cassette pAG1 / promoter GmHsp BRS133 and pAG1 / promoter Gmhsp Soybean varieties BRS133 and pintado (spots) transformed by biovaritics using JF7027 and subjected to drought stress while maintaining embryos on paper filters at 37 ° C. in a greenhouse for 2 hours, 4 hours and 6 hours. The histochemical tests performed on the embryos of. Blue dots indicate a positive reaction. CN-negative control, untransformed embryo; CP-positive control, embryo transformed with plasmid pAG1; S-expression cassette pAG1 / promoter Gmhsp Sensitive embryos transformed with BRS133; R-expression cassette pAG1 / promoter Gmhsp Resistant embryos transformed with JF7027. Sensitive to cyst nematode Javanica, expression cassette pAG1 / promoter GmHsp BRS133 and pAG1 / promoter Gmhsp Shown is a histochemical study performed on embryos of soybean cultivar BRS133 transformed with biovaritics using JF7027 and _{subjected to biological stress by inoculation of 2000-3000 J 2 / mL.} Samples remained in contact with the pathogen for 24 hours, 48 hours and 72 hours. Blue dots indicate a positive reaction. CN-negative control, untransformed embryo; CP-positive control, embryo transformed with plasmid pAG1; S-expression cassette pAG1 / promoter Gmhsp Sensitive embryos transformed with BRS133; R-expression cassette pAG1 / promoter Gmhsp Resistant embryos transformed with JF7027. Sensitive to cyst nematode Javanica, expression cassette pAG1 / promoter GmHsp BRS133 and pAG1 / promoter Gmhsp The histochemical tests performed on embryos of the soybean variety pintado (spots) transformed with biovaritics using JF7027 and _{subjected to biological stress by inoculation of 2000-3000 J 2 / mL are shown.} Samples remained in contact with the pathogen for 24 hours, 48 hours and 72 hours. Blue dots indicate a positive reaction. CN-negative control, untransformed embryo; CP-positive control, embryo transformed with plasmid pAG1; S-expression cassette pAG1 / promoter Gmhsp Sensitive embryos transformed with BRS133; R-expression cassette pAG1 / promoter Gmhsp Resistant embryos transformed with JF7027.

The present invention relates to the use _{of AT (n)} inserts in promoter elements to regulate the expression level of coding sequences in plants. Among resistant and susceptible individuals in the population, the expression levels of heat shock protein (Gmhsp17.6-L) were compared and the maximum expression levels per quantitative PCR were present in individuals containing the maximum AT inserts in the promoter region. Was. To transform soybean embryos, a gene expression cassette containing the promoter region of the gene was constructed with different numbers of AT inserts fused to the GUS protein. According to the experimental results of the present invention, the expression level of certain genes is artificially informed within certain restrictions by inserting _{AT (n) repeated bases into the promoter region via genetic engineering tools.} It is suggested that it could be controlled (upstream) or down (downstream). Therefore, the technique developed in the present invention _{allows regulation of gene expression levels, provided that AT (n)} repeat inserts are inserted at specific points within the sequence of the promoter region.

Based on previous studies attempting to identify molecular markers for use in the supported selection of nematode meloidgine-jawanica-resistant soybean genotypes, the F-binding microsatellite marker SOYHSP176 was added to the tested population. It was highly significant, explaining the characteristic number of cysts in the roots, showing 43% phenotypic variation and 70% of that variation on the entry scale method. It is important to note that the fragments amplified by this marker are present only in susceptible individuals and not in resistant individuals.

This marker was sequenced and then searched for similarity to other sequences of the nucleic acid deposited in the gene bank, which sequence is high with the promoter region of the low molecular weight heat shock protein (Gmhsp17.6-L). It showed homology, was found in soybeans, and was deposited at GenBank (receipt number: M11317). Using the complete sequence of this gene available in GenBank, a new primer pair was designed to amplify fragments in both genotypes.

This strategy was successful and fragments were obtained in both susceptible and resistant individuals. All of the obtained fragments were cloned and sequenced and showed homology to the same promoter region of the gene Gmhsp17.6-L. However, sequence alignment, when compared to susceptible plants, resistant plants had larger inserts of microsatellite in the promoter region of the gene, characterized by _{AT (n) repetition.} In this way, fragments of resistant parent PI595099 origin showed further repetition of ATs 15 and 13 when compared to the sequence of gene Gmhsp17.6-L available in GenBank. On the other hand, the fragment of susceptibility parent BRS133 origin has 6 fewer AT repetitions than the sequences available in GenBank. These results were repeated for resistant and susceptible individuals in the population resulting from this crossbreed, which was also sequenced.

Alignment also showed that all of the amplified fragments had sequences complementary to the primers of the microsatellite marker SOYHSP176. If this is achieved, the next question arises: did amplification not occur in early tolerant plants, even if the annealing regions of the microsatellite primers were present with a high probability? One possibility is more AT repetitions in resistant individuals, which gives the formation of certain tertiary structures, such as hairpins, making it difficult for primers to annealing DNA polymerase during the amplification process. In conclusion, they may have inhibited the formation of amplicon in these plants.

Insertion of microsatellite within the promoter region of this gene was thought to deactivate it in susceptible plants, as the marker SOYHSP176 was amplified only in these individuals. However, studies show that all samples showed insertion of microsatellite, which is probably the number of AT repeats within the promoter region that may interfere with the soybean response to C. elegans. The presence of more or less repetitive sequences into the promoter region of a gene may inactivate, activate, or alter its expression level the protein regulated by this promoter.

According to the literature, one of the main functions of these AT-elements, which are repeated in the regulation of heat shock protein gene expression, is to facilitate access of RNA polymerase to the transcription start site, and in addition, to transcription. Eliminating histones to promote the binding of heat shock proteins involved in induction. According to the literature, heat shock proteins act as molecular sheperons in the cell to establish nascent polypeptide chains, assist in their correct folding, reestablish proteins and / or denatured membranes, and further signal transduction. Plays an important role in.

Therefore, the length of insertion of the gene Gmhsp17.6-L into the promoter region is M.I. A ribonuclease protection assay was used to investigate the hypothesis of altering the expression level of the protein Gmhsp17.6-L in resistant and susceptible individuals affected by Javanica. The results showed that all individuals expressed the protein regardless of resistance or susceptibility, and regardless of treatment, with or without nematode inoculation. Due to the biological function of the AT sequence in gene regulation and the differences that exist between individuals regarding the number of repetitions within the promoter, the ribonuclease protection assay was thought to show the expression chain of the gene Gmhsp17.6-L in different individuals. Note that the results, in theory, showed that there was no difference in the expression of this gene in response to infection by nematodes, at least not at detectable levels by the techniques used.

In an attempt to confirm these results, a new study was devised using real-time PCR (RT-PCR), an accurate and precise technique for quantifying gene expression levels. Lines to parent BRS133 (susceptibility) and PI595099 (tolerance), as well as individuals in the susceptible population (256-S, 259-S and 266-S) and individuals in the resistant population (JF7002, JF7027 and JF7056) from this crossbreeding. Inoculated with insects. Roots of treatments inoculated and uninoculated with Meloid Gine Javanica were recovered for RNA extraction and total cDNA synthesis was then used in the RT-PCR reaction for relative quantification.

The results showed that all resistant parental individuals PI595099, the populations JF7002, JF7027 and JF7056, exhibited higher expression of the gene Gmhsp17.6-L inoculated with cyst nematodes. These individuals in this population are the same as showing _{higher AT (n)} repetitions within the promoter region of the gene in sequencing. Our hypothesis is that when infected by C. elegans, certain types that interact with the AT-rich region of the promoter of the resistant plant gene Gmhsp17.6-L, which activates its transcription with intracellular chaperone function. It is to generate a factor. In plants sensitive to the interaction of factors such as the AT region, it does not occur in such a violent manner, which indicates that the expression level of the protein Gmhsp17.6-L is lower in plants sensitive to nematodes. means.

Through the techniques of the present invention, it is possible to control the expression levels of different genes, including AT inserts of different sizes at specific points in the promoter region. It also makes it possible to develop GM plants that overexpress or underexpress genes that are present in the promoter region in proportion to the size of AT insertion.

The biomaterial construct was generated using the parent PI595099 and BRS133 of the soybean genotype, which are resistant and susceptible to the root-knot nematode Meloidogyne javanica, respectively. From this high distribution, individuals 256-S, 259-S and 266-S in the sensitive population and individuals JF7002, JF7027 and JF7056 in the resistant population were selected in the F4 generation and used in this test.

Also, sensitive and is cultivar Pintado (Pintado) The nematodes are constructed from different samples, genes Gmhsp17.6-L (GenBank accession number: M11317) with an expression cassette comprising different AT _{(n) is} in the promoter region of the It was used via particle collision in the soybean embryo transformation step.

Example 1: Isolation and characterization of the promoter and complete gene Initially, using the complete sequence of the gene Gmhsp17.6-L obtained from the initial sequencing of the amplified fragment of the sensitive material using the microsatellite marker SOYHSP176 primer. Given that only sensitive genotypes amplified the microsatellite marker SOYHSP176, a novel primer set was designed to obtain bands for all genotypes under analysis (FIG. 1).

Primer (pSoyHsp Alle I F 5'CAC CGC GGT G GAA TTC TGA AAT TGG GTC TTT TTG3'; pSoyHsp NcoIR 5'CCA TGG AAT GGG GAC ACT CGA GGT ATT3') was synthesized at the initiation of the promoter region of the gene Gmhsp17.6-L and labeled for later cloning. FIG. 2 shows the primer annealing site in the sequence of the gene Gmhsp17.6-L available in GenBank.

Newer primer set (pSoyHSP) PstI F 5'GGG CTG CAG GAA TTC TGA AAT TGG GTC TTT TTG3'; SoyHSPCL R 5'CCC CCC GGG TTA ACC AGA GAT TTC TAT AGC CT3') was designed, which amplifies all genes from the start to the stop codon of the promoter in the coding region, and is a root-knot nematode. This was to confirm the presence of other differences in the gene sequence, other than _{the promoter-sensible AT (n)} insert between genotypes that are sensitive and resistant to javanica. FIG. 3 shows the primer annealing sites in the sequence of the gene Gmhsp17.6-L, available in GenBank, designed to amplify and sequence the complete gene.

Genomic DNA from seeds was extracted from all analyzed samples. Approximately 50 mg of seeds from each analyzed sample were sliced into flakes with a razor blade, collected in 1.5 mL microtubes and added 300 μL of extraction buffer. The material was ground and an additional 700 μL of extraction buffer was added. Extraction buffers were prepared according to the protocols shown in the table below:
Table 1: Reagents used to prepare genomic DNA extraction buffer:

The solution was homogenized using vortex for 30 seconds to 1 minute and centrifuged at 16,000 xg for 4 minutes at room temperature. The suspension was transferred to a new 1.5 mL tube, provided for a new centrifugation step under the same conditions as last time, and transferred to a new 1.5 mL tube.

To eliminate protein, 0.1 mg of Proteinase K and 0.01 mM CaCl ₂ were added to the sample and incubated in a water bath at 37 ° C. for 90 minutes. 900 μL of cold isopropanol was added, and after incubation for 2 minutes, centrifugation was performed at 16,000 × g for 7 minutes. The supernatant was discarded and the pellet was dried for 90 minutes. Resuspend the pellet in 300 μL TE buffer (Tris-HCl 1M, pH 8.0, EDTA 0.5M, pH 8.0) containing 40 μg / μL RNase A and bathe again to eliminate RNA. Samples were incubated in 37 ° C. for 90 minutes. A new precipitation step is then performed, and finally the DNA pellet is resuspended in 300 μL TE buffer and quantified using a spectropolarizer to determine its quality and completeness on a 0.8% agarose gel. confirmed.

To amplify the promoter region and complete gene sequence from all resistant and susceptible samples, a PCR reaction was performed using a Perkin Elmer 9600 thermocycler with 30 ng of template DNA, 10 × reaction buffer (100 mM Tris-HCl). It is composed of pH 8.3, 500 mM KCl, and 400 μL of ultrapure water), 1.5 mM MgCl2, 1.3 mM dNTP, 1 U of Taq DNA polymerase, and 5 μM of each F and R primer. It was completed with a final volume of 10 μL.

The thermodynamic cycle program used to amplify the DNA sample consists of: an initial denaturation step at 94 ° C. for 7 minutes, followed by denaturation at 94 ° C. for 1 minute, annealing at 60 ° C. for 1 minute, And 30 cycles of elongation for 2 minutes at 72 ° C. The final cycle was performed at 72 ° C. for 7 minutes. The amplification product is separated on a 1% agarose gel and used with 1 × TBE buffer (108 g Tris base, 55 g boronic acid, 40 mL EDTA 0.5M pH 8.0, and distilled water, complete with a final volume of 1 L). And stained with ethidium bromide (10 mg / mL). Images were obtained using the Kodak Digital DC290 system.

Amplified fragments (FIGS. 4 and 5) were extracted using the PureLink ™ Quick Gel Extension (Invitrogen) kit according to the manufacturer's instructions. After purifying the DNA, it was stored at −20 ° C. Next, the DNA was quantified and the ligation reaction between the fragment and the plasmid vector was performed using TOPO® TA cloning kit (Invitrogen). The ligation reaction consisted of: 2 μL ultrapure water; 1 μL salt solution (dilution of 7 μL salt solution: 21 μL ultrapure water); 1 μL TOPO® vector and 2 μL (about 10 ng) purified band. .. Samples were incubated at room temperature for 5 minutes.

The resulting vector was used for E. coli (DH10B strain) cell transformation through electroporation. Electrocompetent cells were prepared and colonies isolated from glycerol stocks were obtained through plate streaking. The five single colonies were then cultured overnight at 37 ° C. at 200 rpm in 10 mL of LB medium-Luria Bertani (a 20 g mixture of tryptophan, yeast extract and NaOH in 1 L of distilled water). (Pre-inoculation) 5 mL of pre-inoculation was added to 500 mL of LB medium rotated 300 rpm at 37 ° C. until OD500 reached 0.5-0.7. After this period, cells were incubated on ice for 20 minutes and transferred to a 250 mL centrifuge tube pre-chilled with ice in a sterile condition. Centrifugation at 4,278 xg for 15 minutes at 4 ° C. was performed, the supernatant was discarded and the pellet was resuspended in 500 mL of chilled 10% glycerol. New centrifugation was performed under the same conditions, the supernatant was discarded and the pellet was resuspended in 250 mL of chilled 10% glycerol. Centrifugation was performed again and the pellet was resuspended in 20 mL of chilled 10% glycerol. The solution was transferred to a 50 mL sterile tube and centrifuged at 3997 xg for 15 minutes at 4 ° C. As a final step, the pellet was resuspended in 1 mL-2 mL of chilled 10% glycerol. Electrocompetent cells were dispensed into 500 μL microtubes and stored in a -80 ° C freezer.

Electroporation was performed with a MicroPulser (BioRad) electroporator set to 2.5 kV, 25 μF and a pulse controller set to 200 or 400 ohms. 2 μL of ligation reactants and 100 μL of electrocompetent cells were used. Cell rescue in 15 mL sterile Falcon tubing, 1 mL SOC medium (0.5 g yeast extract, 2 g tryptophan, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl ₂ , 10 mM _{sulfonyl 4} , 20 mM glucose, and 96 mM. The bacterial suspension in distilled water) was cultured and incubated at 37 ° C. for 1 hour at 200 rpm. After this period, 300 μL of electroporated cells supplemented ampicillin (100 μg / 30 mL) with IPTG / X-Gal (50 μl of IPTG-isopropyl-β-thiogalactopyranoside 0.1 M, and 10 μL of X. It was seeded in solid LB medium containing −Gal−−bromo-4-chloro-3-indrill-β-D-galactoside (50 mg / mL) / plate). The plates were incubated at 37 ° C. overnight for colony growth. Recombination colonies are selected using the lacZ system, with recombinant colonies showing white colonies and non-recombinant colonies showing blue colonies. This is due to the resulting product of the reaction of the lacZ gene product with the substrate (X-Gal).

Next, to confirm the cloning and size of the fragments, the plasmid DNA extracts were 1 mL CG medium-cycle glow (4 g commercial mixture to 100 mL water) and 100 μL / mL ampicillin (50 mL CG medium). It was performed by inoculation of a single white colony in 96-well microplates containing 50 μL of ampicillin (100 mg / mL). The plate was sealed and the material was incubated at 37 ° C. at 320 rpm for 22 hours.

Prior to proceeding with plasmid extraction, a permanent culture of growing bacteria was obtained and 75 ml of overnight grown CG medium and 50% sterile glycerol were contained in a sterile plate. This culture was stored in a super freezer at −80 ° C. and used as a stock.

The miniprep was continued and the microplates were centrifuged for 6 minutes at 1,310 xg, 10 ° C. to obtain cell deposits. The supernatant was discarded and the plate was quickly inverted on absorbent paper. Next, 200 μL of GET (20% glucose, 0.5 M EDTA pH 8.0, 1 M Tris-HCl pH 7.4 and water to fill 500 mL) was added to each plant, the plate was sealed and vortexed for 2 minutes. The cells were resuspended by vigorously shaking with. A fresh centrifuge at 1,310 xg, 10 ° C. was performed for 9 minutes, the supernatant was discarded, and the plate was quickly inverted on absorbent paper and dried.

65 μL of GET containing RNase A (10 mg / mL) was added to each well of the microplate. All material was resuspended using vortex and transferred to a "U" bottom plate. Next, 65 μL of NaOH 0.2N / SDS 1% (0.5 mL NaOH 4M + 1 mL SDS 10% + ultrapure water, 10 mL volume) was added to each well. This solution was prepared just prior to the step in which it was used. The plates were sealed; the materials were mixed by inversion for 5-10 minutes and incubated for 10 minutes at room temperature. Rapid centrifugation was performed for a few seconds until no solution remained on the adhesive seal.

To each well, 60 mL of 3M potassium acetate (KOAc) stored at 4 ° C. was added, the plates were sealed, the materials were mixed by inversion 10 times and the incubation for 10 minutes was performed on ice. Centrifuge at 1,310 xg, 4 ° C. for 15 minutes, transfer 80 μL of supernatant to a Millipore® (MAGV N22) plate and preliminarily on top of a 250 μL “V” bottom polypropylene microplate. Fixed. The plate was centrifuged for 5 minutes at 1,310 xg, 4 ° C., or until the entire volume was transferred to the bottom plate (V bottom). The Millipore® plate was removed, discarded and 80 μL of isopropanol was added to the filtered solution transferred to the “V” bottom plate.

The "V" bottom plate was sealed with an alcohol resistant adhesive and the materials were inverted and mixed. Centrifugation at 1,310 × g, 4 ° C. was performed for 45 minutes, the supernatant was discarded by inverting the plate, and 150 μL of cold 70% ethanol was added to each sample. The plate was again centrifuged for 5 minutes at 1,310 xg, 4 ° C., the supernatant was discarded, then the plate was inverted on absorbent paper and quickly spun down at 82 xg, 4 ° C. The plate was covered with a paper towel, dried at room temperature for 60 minutes, and finally the DNA was resuspended in 30 μL of ultrapure water. The plates were sealed and incubated overnight at room temperature for complete elution of plasmid DNA. After this period of time, the plate containing the plasmid DNA was stored in a freezer at −20 ° C.

Samples were subjected to digestion reactions with restriction enzymes to confirm the presence and size of inserts before being prepared for sequencing. The digestion reaction is composed of 1.5 μL of reagent 3 enzyme buffer; 0.5 μL of EcoRI (10 U / μL); 5 μL of plasmid DNA (about 10 ng) and 8 μL of ultrapure water, and is incubated at 37 ° C. for 2 hours. bottom. After this period of time, the aliquots of the reactants were subjected to 1% agarose gel electrophoresis.

Fragments are sequenced by an ABI Prism 3100 (Applied Biosystems) capillary sequencer, in which case an ABI Prism BigDye terminator cycle sequencing (Applied Biosystems) kit is used based on both DNA double-stranded orientations. Different sequencing experiments, M13R and F primers were used. For the sequencing reaction, 1.5 μL of DNA from each sample was placed on the plate and 8.5 μL of the reaction mixture prepared according to the protocols listed in the table below was used.

Table 2: Reagents used to prepare the reaction mixture used for the sequencing reaction

The sequencing reaction was performed on an Applied Biosystems thermocycler under the following conditions: 96 ° C. for 2 minutes; 96 ° C. for 15 seconds, 50 ° C. for 15 seconds, and 60 ° C. for 4 minutes for 30 cycles. Finally, the reaction for DNA precipitation was carried out by adding 80 μL of 75% isopropanol to each sample, then incubating for 15 minutes at room temperature, shading and 45 minutes at 1,310 xg. Centrifugation was performed.

The supernatant was discarded, the pellet was washed with 100 μL of 70% ethanol, and then a 1,310 × g centrifugation step was performed for 15 minutes. The supernatant was discarded, the pellet was dried at room temperature, and the pellet was dried. For sample application on a sequencing machine, the pellet was resuspended in 10 μL hi-formamide and then incubated for use in a thermocycler at 95 ° C. for 5 minutes to cause DNA denaturation. To avoid DNA reannealing, the plates were immediately transferred to ice and then placed on a sequencing machine.

After sequencing the promoter region and the complete gene region, a sequence database search of the biological sequence was performed to verify the similarity between the obtained fragment and the known DNA and protein sequences (Fig. 6). To achieve this, the programs BLASTn and BLSATx (Altschul et al. (1997), Nucleic Acids Res. 25: 3389-3402) were used. Nucleotide sequences (FIGS. 7 and 8) and amino acid sequences (FIG. 9) are also aligned using suitable software, such as Vector NTI Advanced 10.0.1 (Invitrogen Corp.), BioEdit Sequence Identor and Bluestal W. I went.

Example 2: Regulatory Study of Gene Expression-RPA
Parent genotypes PI595099 (resistant) and BRS133 (susceptibility) were selected for RPA experiments. A primer set was designed for the coding sequence from the complete sequence of the heat shock protein Gmhsp17.6-L (GenBank accession number: M11317) present in soybean (RPA2). F 5'GAC ATC ATC AAA CAA GAG AA3' and RPA2 R 5'TCT CTC CGC TAA TCT GAA3').

The seed DNA extract from the parent sample analyzed, amplification of the fragment through PCR, cloning and sequencing of the product produced by PCR was performed according to the protocol previously detailed in the previous section.

Total RNA from parent PI595099 (resistant) and BRS133 (susceptibility) was extracted from soybean roots subjected to two different treatments (M. Javanica nematode egg inoculation and non-inoculation). Extraction was performed using a Trizol reagent (Life Technology) kit. Roots are ground in liquid nitrogen using a mortar and pestle, transferred to an autoclaved Falcon tube and stored in a draft, which contains 20 mL of trizol. After homozygation, the samples were incubated for 5 minutes at room temperature (15-30 ° C.). 0.2 mL of chloroform was added to each of the 1 mL of trizol, and after vigorous stirring for 15 seconds, the solution was incubated at room temperature (15-30 ° C.) for an additional 3 minutes. Next, a centrifugal separation step of 12.240 × g for 15 minutes at 4 ° C. was performed. The liquid phase containing RNA was transferred to a new falcon tube and precipitated with 0.5 mL isopropanol for each of the first 1 mL of trizol used. The solution was incubated at room temperature (15-30 ° C.) for 10 minutes. 12.240 × g, 10 minutes, a new centrifugation step at 4 ° C. was performed. The pellet formed at the bottom of the tube contains precipitated RNA. The total supernatant was removed and the RNA pellet was washed with 75% ethanol (1 mL ethanol for each 1 mL of trizol used). The solution was centrifuged at 4.285 × g for 5 minutes at 4 ° C., and the total supernatant (ethanol) was removed again. To dissolve the pellets, 400 μL of DEPC water was added (if necessary, the temperature was raised to 50-60 ° C. to dissolve the pellets). After extraction, total RNA was quantified on a spectropolarizer; its integrity was verified on a 2% agarose gel and finally stored in a super freezer at -80 ° C.

Initially, the plasmid containing the fragment of interest was linearized. The reaction containing 30.6 μL of DEPC water, 4 μL of buffer, 1 μg of purified plasmid, 0.4 μL of BSA, and 1 μL of the restriction enzyme ApaI was incubated at 37 ° C. for 2 hours in a thermocycler. After this period of time, the plasmid was purified with 1 volume of phenol, mixed with vortex for 2 minutes and subjected to a centrifuge step at 11.750 xg for 10 minutes at 4 ° C. The aqueous phase was then recovered and ammonium acetate (NH ₄ OAc) was added to bring the final concentration to 0.5M. Three volumes of cold 95% ethanol were added and the solution was incubated at −20 ° C. for 1 hour. A new centrifugation step of 11.750 xg, 4 ° C., 15 minutes was performed and the supernatant was carefully discarded. The tube was opened; the pellet was dried for 5 minutes, then resuspended in 30 μL of DEPC water and stored in the freezer at −20 ° C.

Obtaining a Probe The transcription reaction to obtain a probe was performed using the MAXISscript ™ In vitro Transcription (Ambion Inc.) kit. RNA polymerase T7 and P ³² -labeled phosphate dideoxynucleotide (CTP) were used. First, all kit reagents were thawed and maintained on ice. However, Transcription buffer (registered trademark) that must be stored at room temperature is excluded. The transcription reaction using a total final volume of 20 μL was carried out in 1.5 mL microtubes at room temperature according to the protocols described in the table below.

Table 3: Reagents used for transcription reaction of radioactive probe

All reagents were then gently mixed in microtubes and the reaction was transferred once at 37 ° C. and incubated using a thermocycler. 2U DNase I RNase-free was added and the reaction was incubated for 15 minutes at 37 ° C. Add 1 volume of gel-loading buffer (95% formamide, 0.025% bromophenol blue, 0.025% xylene cyanol, 0.5 mM EDTA, 0.025 SDS) to the reaction and tubing for 3-5 minutes. , 85-95 ° C., and all samples were applied on a 5% polyacrylamide gel, urea 8M, 1 × TBE buffer.

A gel with a final volume of 25 mL was prepared using 12.01 g of urea, 3.12 mL of 40% acrylamide: bis-acrylamide (19: 1) and 2.5 mL of 10 × TBE. A small amount of water was added further to avoid exceeding the final volume of the gel. To dissolve the urea, the solution was maintained in a magnetic agitation system equipped with a heater, then cooled; transferred to a cylinder and the final volume of gel was filled with DEPC water. Next, 12.5 μL of TEMED was added, and finally 156.2 μL of 10% ammonium persulfate was added.

The solution was immediately applied to a glass plate, a well comb was placed and polymerization was carried out without moving the plate. Pretreatment of these materials can be performed, consisting of washing the glass plate and the electrophoresis chamber and treating with RNase AWAY® (Invitrogen-Life Technology). On a smaller plate, 1-3 mL Repel can be provided in the draft and the plate can be dried for 5-10 minutes. This treatment facilitates gel removal after electrophoresis.

Prior to electrophoresis, the plates were washed to remove excess urea, and after removal of the wellcoms, the wells were washed with running buffer with the help of a syringe. A 20-30 minute pre-drive of the gel using a predetermined voltage was performed on the chamber, after which the use was applied. Depending on the size of the probe, about 2 hours of electrophoresis was performed at 200-300 volts.

Only the full-length probe was gel-purified. To achieve this, after electrophoresis, the plate was opened, the gel was covered with a plastic film, and the autoradiography film marked to guide band position identification was exposed for about 1 hour. The film was then developed by immersing it in a developer in a dark room for 5 minutes to remove excess, soaking in water for about 3 minutes for washing and finally in a fixed solution for 5 minutes. The film was then immersed in water and air dried. The film and gel were lined up to locate the full-length transcript. The target area of the gel was removed and transferred to a microtube containing 350 mL of elution buffer (0.5 M ammonium acetate, 1 mM EDTA, 2% SDS). The tube containing the probe was incubated overnight at 4 ° C. to maximize probe regeneration and store it at 20 ° C. until hybridization. The kit for labeling the probe provided a positive control for monitoring its quality.

A probe was synthesized from a control DNA containing an insert of a 250 bp rat β-actin gene and a linearized pTRIPLEscript plasmid and hybridized to rat liver total RNA. They were also given to the kit. The two control tubes contained only yeast total RNA and were used to verify the activity of the RNase enzyme. In this way, the enzyme positive control tube contained only RNase and buffer, while the negative control tube contained only enzyme-free buffer.

Sample Hybridization and Digestion The Ribonuclease Protection Assay (RPA) was performed using the HySpeed ™ RPA-Hygh-Speed Hybridization Ribonuclease Protection Assay (Ambion Inc.) kit. For each microtube, labeled probe was added to total RNA (20 mg) (high specific activity, 250 nt of about 100-800 pg or 2-8 x 104 cpm of 1-10 fmol). In addition, 30 mg of total yeast RNA was added to the sample to obtain a final 50 mg / sample. Two other enzyme control tubes containing 10 mL of total yeast RNA (50 mg) were obtained, one containing rat liver total RNA as a probe-positive control. To coprecipitate the probe + sample, 0.5 M NH ₄ OAc and 3 volumes of cold 95% ethanol were added, homozygated and the tubes were incubated for 15 minutes at −20 ° C. Centrifugation was performed at 4 ° C. for 5 minutes at maximum speed (minimum 8.160 xg). The supernatant was removed and the pellet was dried. 10 mL of HySpeed Hybridization Buffer® (pre-heated to 95 ° C.) was added to each sample and the tube was immediately incubated at 95 ° C. (water bath or thermocycler). To dissolve the pellet, the sample was vortexed for a few seconds and returned to 95 ° C. After the pellet was completely dissolved, the tubes were incubated at 95 ° C for 3 minutes and at 68 ° C for 10 minutes. The temperature must be maintained at 68 ° C. and the moving step must be no more than 30 seconds. First, HySpeed RNase digestion buffer® is thawed and a mixture of RNase A / T1 and buffer (1: 100 dilution-1 mL RNase in 99 mL buffer) is applied to each sample in an appropriate amount of 100 mL. The volume was adjusted and stored at room temperature. Add 100 mL of mixed RNase A / T1 + buffer to each sample and control tube containing only total yeast RNA, add 100 mL of enzyme-free HySpeed RNase Digestion® buffer to one tube and add the enzyme. The complete mixture containing was added to the other control tube. Samples were vortexed and incubated at 37 ° C. for 30 minutes for digestion. 150 mL of HySpeed Incubation / Precipitation Buffer® was added to the sample, vortexed quickly and the tubes were incubated for 15 minutes in a -20 ° C freezer. After digestion, the sample was centrifuged for 15 minutes at a maximum speed of 4 ° C., the supernatant was removed and resuspended in gel loading buffer (usually 9-10 mL volume). After pipette homozygation, the tube was heated to 90-95 ° C. for 3-4 minutes and immediately transferred to ice to avoid regeneration. Samples were applied to 5% polyacrylamide gel, 8M urea, diluted in 1x TBE buffer and electrophoresed at 200-300 volts for about 2 hours to separate protected fragments. After electrophoresis, standard techniques were used for film exposure and development detailed in the protocol described above (FIG. 10).

Example 3: Gene expression-RT-qPCR-Experiment set in a greenhouse For the expression test of the promoter of the gene Gmhsp17.6-L, the materials PI595099, BRS133, 256-S, 259- were used using RT-PCR technology. The seeds from which S, 266-S, JF7002, JF7027 and JF7056 were derived were sown on germination paper in a growth chamber and after 8 days the seedlings were transferred to a plastic container. In the greenhouse, these seedlings were infected with the larvae Meloid Gine Javanica at the stage of outbreak J2 infection. The nematode population used was obtained from soybean plants from the cultivar Doko. The nematode egg extract from the root was obtained by grinding the material with a blender for 30 seconds in a 0.5% hypochlorite solution. The roots were washed in water and eggs were collected using a miniscreen with exclusion size 500. The free egg suspension was then transferred to a hatching chamber set at 26 ° C. and Javanica (J2) was collected and stored in a refrigerator every 24 hours. Javanica J2 was quantified in the Peters chamber. Inoculate 664 J2 / ml (per plant) with the help of a pipette and bulk the roots from 3 soybean plants of each of the 8 materials under test 1st, 3rd and 6th days after inoculation. And those inoculated with Javanica and those not inoculated (control treatment) were transferred to liquid nitrogen. Roots were stored in a super freezer (-80 ° C) until the start of the RNA extraction experiment.

RT-qPCR primer design Primer used for real-time PCR (SoyHspPSC F 5'GCT GTG TGT CAT TGT CAT CGA A3'; SoyHspPSC R5'CAC GGT CTA TTT CTT GCC TAC ATC3') uses the sequence pos stop codon (TAA-nucleotide position 884) of the Gmhsp17.6-L gene (GenBank accession number: M11317) to Primer Express 2.0 (Applied). Designed with the Biosystems) program. These primers were used to amplify a fragment of about 80 bp. The selected parameters applied to the Primer Express program to design the primers were an amplicon length of 50 bp to 150 bp (120 bp is recommended for RT-PCR), CG content of 40% to 60%, up to a row. The maximum difference in Tm between 4 G bases, 58 ° C. to 60 ° C. primer Tm (melting temperature), and F and R primers is 1 ° C., and up to 4 identical bases in a row should be avoided. The program OMIGA was then used to analyze the formation of primer dimers and the minimal presence of 6 free bases at the 3'end was observed.

Obtaining Total RNA for cDNA Synthesis Total RNA was extracted using Trizol reagent (Invitrogen-Life Technology) for real-time PCR experiments. First, 1 mL of trizol / sample was dispensed into a falcon tube and heated to 55 ° C. The plant tissue of the sample was homogenized in liquid nitrogen and 0.1 g was dispensed into a tube maintained in nitrogen. Then 1 mL of heated trizol was added to the sample, vortexed for 1 minute, spun down quickly and incubated for 2 minutes at 55 ° C. The sample was centrifuged at 16.000 × g for 20 minutes at 4 ° C., the supernatant was transferred to a tube containing 200 μL of chloroform, and the residue was discarded. The sample was shaken and incubated at room temperature (22-25 ° C.) for 2 minutes, then centrifuged at 16.000 × g for 30 minutes at 4 ° C. The supernatant was transferred to a new tube and 1/3 volume of 8M LiCl was added. After shaking, the tubes were incubated in a freezer at −80 ° C. for 1 hour. To thaw the solution, the tube was maintained in a water bath at 40 ° C. for 1-3 minutes and centrifuged at 16.000 × g, 4 ° C. for 30 minutes. 400 μL of 75% ethanol was added to each sample to remove the supernatant and discard to avoid interfering with the pellet containing RNA, and the tube was gently inverted. A 16.000 × g centrifugation step was performed at 4 ° C. for 5 minutes to remove the supernatant and discard completely. 100 μL of ultrapure water was added to the pellet and the tube was gently tapped to dissolve the pellet (if the pellet does not dissolve quickly, another 100 μL of ultrapure water can be added, but in the next step. The volume of sodium acetate and isopropanol should be doubled). Next, 10 μL of sodium acetate (3M) and 100 μL of isopropanol were added and the tube was gently inverted. Samples were incubated at −80 ° C. for 30 minutes, transferred to a water bath at 37 ° C. for 1-3 minutes, and centrifuged at 16.000 × g, 4 ° C. for 15 minutes. The supernatant was removed, 400 μL of 70% ethanol was added, and the tube was once again centrifuged at 16.000 × g at 4 ° C. for 15 minutes. The supernatant was removed, discarded and the pellet was dried on the bench for 10 minutes. Finally, 50 μL of ultrapure water (or 100 μL if the pellets did not dissolve immediately) was added and the pellets and solution were heated at 37 ° C. for 10 minutes to promote dissolution of the pellets. RNA was quantified and stored in a freezer at -80 ° C. For the reverse transcription reaction, RNA was diluted and cDNA synthesis was performed using reverse transcriptase (Moloney murine leukemia virus: M-MLV). In this way, 1.5 mg of total RNA was dispensed into microtubes, DEPC water was added to a final volume of 9 mL, 6 mM random primers were added to the reaction, and then incubated at 80 ° C. for 3 minutes. After this period, the tube was cooled on ice and 14 mL of the mixture was added to the sample. This mixture was prepared according to the protocols listed in the table below.

Table 4: Reagents used to prepare the mixture for cDNA synthesis Volume 5 per reaction x 1st chain buffer 6 mL dNTP (2.5 mM) 4 mL
DTT (0.1M) 2mL
Reverse transcriptase 2mL

The reaction was incubated at 37 ° C. for 1 hour using a thermocycler, followed by a 10 minute step at 65 ° C. The cDNA was stored at −20 ° C.

RT-qPCR
The PCR reaction uses a Thermal Cycler 7300 real-time system (Applied Biosystem) and a Platinum® SYBER® Green qPCR SuperMix UDG (Invitrogen-Life Technology) kit according to the manufacturer's instructions. I was broken. As recommended by Applied Biosystem, amplification efficiency curves are performed for the target gene Gmhsp17.6-L and the endogenous control gene rRNA 18S (GenBank accession number: X02233.1) (used for sample standardization). rice field. Experimental plates were set up using samples with 3-point measurements for both genes. The curve gives a slope, which is used to calculate the amplification efficiency of the primers and should be similar for both genes and close to 100% (value 1). Amplification reactions for relative quantification were performed using bulk cDNA from 3 days of recovery for each of the analyzed samples. In separate experiments, two real-time PCR experiments were performed for parental use and two for population samples. Parent strains BRS133 and PI595099, and six resulting individuals from susceptible populations, 259-S, 259-S and 266-S, and resistant JF7002, JF7027 and JF7056 were analyzed for nematode inoculation and non-inoculation treatment. Was done. The reaction was performed on a 3-point basis and consisted of 8.0 μL ultrapure water, 0.5 μL ROX, 12.5 μL SYBER® Green qPCR SuperMix UDG and 2 μL bulk DNA (approximately 1.5 μg). .. The cycling parameters of the amplification reaction were as follows: 50 ° C. for 2 minutes; 95 ° C. for 2 minutes; then 95 ° C. for 15 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 30. 45 cycles of seconds. The data was collected in the stretching step (72 ° C.). After concluding the relative quantification, dissociation curves were performed to verify primer dimer formation, nonspecific amplification, possible errors and contamination. Interpretation of the data generated by RT-PCR-was performed using SDS-Sequence Detection System (Applied Biosystem) software.

In these analyses, relative gene expression (RQ) was calculated individually, and as calibration (value 1), non-inoculated samples were used and the samples were compared separately for nematode inoculation and non-inoculation treatments. The RQ value was calculated using the ΔCt method. In this way, the level of gene expression (RQ) is calculated by subtracting the Ct of the target sample for each treatment by the endogenous control Ct, resulting in ΔCt. This value is subtracted from ΔCt of the control sample (calibration-value 1) to obtain the value of ΔΔCt. RQ is obtained through Equation 2-ΔΔCt. Where 2 corresponds to the sum of the target gene efficiencies (100% = 1) and the synthesis of the endogenous control (100% = 1) obtained on the 100% efficiency curve (Live and Schmittgen (2001), Methods 25). : 402-408). The efficiencies of the target and endogenous genes should be close to 100%, but not necessarily, but they may be lower, however, they must be close. Then, in this case, the value 2 of the formula 2-ΔΔCt is replaced by the sum of the efficiencies of the target gene and the endogenous gene. Root hump nematode M. For analysis of parent strains BRS133 and PI595099 in inoculated and non-inoculated treatment with Javanica, analysis of susceptible groups (256-S, 259-S and 266-S) using uninoculated sample PI595099 as calibration (value 1X). And for resistant populations (JF7002, JF2027 and JF7056), uninoculated sensitive samples 256-S were used as calibration samples. This material was selected due to the fact that it showed a low number of AT (n) inserts in sequencing the promoter region of the Gmhsp17.6-L gene.

Example 4: Expression cassette construct containing AT (n) inserts of different sizes in the promoter region of the Gmhsp17.6-L gene A resistant individual has a high number of AT (n) inserts in the promoter region of the Gmhsp17.6-L gene. These individuals, when subjected to RT-PCR, showed the root-knot nematode M. elegans. Once inoculated with Javanica and confirmed to exhibit high levels of this gene expression, the expression cassette is constructed to contain different numbers of AT inserts in the promoter region of the Gmhsp17.6-L gene. Was decided on. The purpose of utilizing these constructs is that these promoters, along with AT (n) inserts, induce the expression of other genes and also respond to those genes, activating or deactivating them by different stresses. Can be evaluated. Cassettes were used to transform nematode-sensitive varieties BRS133 and Pintado-derived soybean embryos using particle collisions. In the resulting construct, the promoter of the Gmhsp17.6-L gene is located before the Gus gene, which has expression via histochemical assays, after exposure of the embryo to different stresses.

Expression cassettes containing the promoter regions of 8 analyzed samples BRS133, PI595099, 256-S, 266-S, JF7002, JF2027 and JF7056 were obtained, for a total of 96 cassettes (12 for each material). rice field. However, only two constructs were used in the next step of this test, which is AT (9), a cassette pAG1 / promoter containing an amplified promoter region from the susceptible parent BRS133 with a smaller number of inserts. Gmhsp Cassette pAG1 / promoter Gmhsp containing amplified promoter regions from individuals from the resistant population JF7027 with BRS133 and AT (32) It was JF7027. The plasmid pAG1 was chosen as a template for constructing an expression cassette. The designed strategy was the removal of the act2 promoter from the actin gene present in the plasmid and the insertion of the susceptible parent strain BRS133 and the amplified promoter from the material to be analyzed, which is an individual of the resistant population JF7027. .. The plasmid also shows the reporter gene Gus, which encodes the β-glucuronidase enzyme, which is allowed by visualization in transgenic embryos through histochemical assays if the transformation process with those cassettes is successful. In addition, the plasmid pAG1 was mutated at position 653 to cause the herbicide Imazapyr (2- [4,5-dihydro-4-methyl-4- (1-methylethyl) -5-oxo-1H-imidazol-2-yl). ] -3-Pyridinecarboxylic acid) (Tu et al. (2004), Weed control plasmids handbook; London: Academic) belongs to the herbicide imidazolinone-derived enzyme acetolactic acid pyruvate lyase that imparts resistance to imidazolinone. Includes a promoter (ahas 5), coding sequence (ahas) and terminator (ahast) of the als / ahas gene encoding (AHAS) -acetolactic acid synthase (ALS). In this way, this gene allows selection of positive plants in herbicide-containing media after transformation. FIG. 13 shows a plasmid map. To develop this strategy, plasmid DNA extraction was performed using the Wizard Plus Maxipreps DNA Purification System (Promega) kit. In this way, single colonies were inoculated into 2-5 mL of LB medium containing the antibiotic ampicillin (0.5 mg) and incubated at 37 ° C. for 8 hours at 200 rpm (pre-inoculation). .. 1 mL of pre-inoculation was transferred to 500 mL of LB / ampicillin medium and incubated at 37 ° C. for 12-20 hours with rotation at 200 rpm. Cells are dispensed into 250 mL tubes, centrifuged at 5.000 xg for 10 minutes at room temperature, the supernatant is discarded, and 15 mL of cell resuspension (50 mM Tris-HCl, pH 7.5, 19 mM EDTA). , 100 μg / mL RNase A) completely resuspended the pellet. 15 mL of cytolytic solution (0.2 M NaOH, 1% SDS) was added, inverted and mixed, and the solution was incubated for 20 minutes at room temperature. Next, 15 mL of neutralized solution (1.32 M potassium acetate, pH 4.8) was added, the solution was inverted and mixed, resulting in the formation of white flakes, the lysate being less viscous. Precipitated material contains genomic DNA, proteins, cell debris and SDS. Centrifugation was performed at 14.000 xg for 15 minutes at room temperature and the supernatant was filtered through a cylinder containing Whatman 1 filter paper. The volume was measured, transferred to a 50 mL centrifuge tube, 0.5 volume of isopropanol at room temperature was added, inverted and mixed. A new centrifugation step was performed at 14.000 xg for 15 minutes at room temperature, the supernatant was discarded and the pellet was resuspended in 2 mL TE buffer. To the solution containing this DNA, 10 mL of Wizard Maxipreps DNA Purified Resin® was added and the resin + DNA mixture was transferred to a Vacuum pump-linked Maxicolum. A vacuum was created and 25 mL of column wash solution (80 mM potassium acetate, 8.3 mM Tris-HCl, pH 7.5, 40 μM EDTA, 55% ethanol) was added and further evacuated. 5 mL of 80% ethanol was applied to the Maximolun and evacuated, after which the whole cough ethanol was passed through the column and evacuated for an additional minute. Next, Maxicolum was transferred to a 50 mL falcon tube and centrifuged at 1.300 xg for 5 minutes. Vacuum was applied for an additional 5 minutes to allow the resin to dry. Finally, Maxicolum was transferred from the kit to the provided tube and 1.5 mL of preheated water (65 ° C-70 ° C) was added to the column. After 1 minute, a centrifugation step (1.300 × g, 5 minutes, room temperature) was performed to elute the DNA. After extraction, plasmid DNA was quantified and its integrity verified on a 1% agarose gel. Restriction enzyme digestion was performed on the act2 promoter excision (-1000 bp). In the first step, pAG1 was subjected to double digestion with the enzymes Pst1 and Not1. This digestion resulted in approximately 5.500 bp (corresponding to the promoter-ahas 5, coding sequence-ahas and terminator-ahast of the als / ahas gene) and 6.500 bp (corresponding to the Gus gene, act2 promoter and plasmid backbone, Ori and Nos). (Corresponding) two fragments were obtained. The digestion reaction consisted of 10 μL of plasmid DNA (approximately 1.5 μg), 2 μL of React 2 buffer, 5.0 μL of Pst1 enzyme (10 U / μL), and 3 μL of ultrapure water. The tubes were incubated at 37 ° C. for 2 hours using a thermal cycler. After this period, another 2 μL of Pst1 enzyme (10 U / μL) was added to the solution and incubated at 37 ° C. for 2 hours using a thermocycler. The reaction with Not1 was carried out in the same microtube, and 3.5 μL of Not1 enzyme (10 U / μL), 1.5 μL of Reat 2 buffer and 2 μL of NaCl (1 M) were added. A fresh incubation at 37 ° C. for 2 hours was followed by an additional 2 μL of Not1 enzyme. The reaction was maintained overnight using a 37 ° C. thermocycler. The pAG1 digested fragments were separated on a 0.8% agarose gel and the gel was extracted using the PureLink ™ Quick Gel Execution (Invitrogen) kit according to the protocol described above. In the second step of digestion, the larger fragment (6.500 pb) was digested with the restriction enzyme BamHI for excision of the act2 promoter. The reaction consisted of 0.5 μL of BamHI enzyme, 15 μL of React 3 buffer, 12 μL of DNA digestion fragment, and 1 μL of ultrapure water. Using a thermocycler, the microtubes were incubated for 2 hours at 37 ° C. After this period, an additional 0.5 μL of BamHI enzyme was added and the reaction was further incubated overnight at 37 ° C. Fragments are separated on a 0.8% agarose gel and larger fragments (corresponding to Gus gene, plasmid DNA, Ori and Nos) are prepared according to the manufacturer's instructions for the PureLink ™ Quick Gel Extraction (Invitrogen) kit. Was purified by gel. To obtain an expression cassette construct containing different amplified promoters from the parent strain BRS133 and resistant individual JF7027, the ligation reaction was 11 μL ultrapure water, 2 μL (about 10 ng) Ahas fragment from each tested sample, 1 μL ( About 10 ng) of Gus fragment, 1 μL (about 10 ng) of purified band from the Gmhsp17.6-L promoter, 4 μL of ligase buffer and 1 μL of T4 ligase enzyme were mixed. The reaction was incubated overnight at 14 ° C. Promoter region fragments from two selected samples are primer pSoyHspPstI F (5'GGG CTG CAG GAA TTC TGA AAT TGG GTC TTT TTG3') and pSoyHspBamHI It was amplified from genomic DNA using R (5'CCC GGA TCC AAT GGG GAC ACT CGA GGT ATT3'). The restriction enzyme sites designed and synthesized in the primers allowed cloning of the Gmhsp17.6-L gene promoter in the correct orientation at the same position where the act2 promoter was previously located. According to the protocol described above, E.I. The colli cells, DH10B strain, were electroporated using a cassette ligation reaction and grown overnight in LB medium at 37 ° C. From the obtained colonies, a series of PCR amplification using specific primers and a digestion reaction using restriction enzymes were performed to verify the successful cloning of the construct. After this step to prove the success that the expression cassette was obtained, for the Ahas gene (654bp) of the pAG1 plasmid, a primer specific for the promoter region of the Gmhsp17.6-L gene (Ahas1). F5'ACT AGA GAT TCC AGC GTC AC3'; Ahas2 For R5'GTG GCT ATA CAG ATA CCT GG3') and Nos Terminator (Nos1) F5'GAA TCC TGT TGC CGG TCT TG3'; Nos3 PCR reactions using R5'TTA TCC TAG TTT GCG CGC TA3') were performed using all the obtained samples. FIGS. 14, 15 and 16 show the amplification of the promoter region of Gmhsp17.6-L, part of Ahas and part of Nos region, respectively, of each of the 12 samples of the obtained expression cassette.

Example 5: Study of transient expression or GUS (β-glucuronidase) activity Acquisition of transgenic plants through particle collisions Expression cassette pAG1 / promoter GmHSP BRS133 and pAG1 / promoter GmHSP To obtain transgenic plants transformed with JF7027, plasmid DNA extraction was first performed according to the protocol described above. This technique yielded a sufficient amount of plasmid DNA for use in biovaritics, gene gun methods, or transformation techniques via particle collisions. In this technique, the introduction of genetic material into the plant is carried out with fine particles and is usually made of gold or tungsten with a diameter of 0.4-0.2 mm, coated with endogenous DNA. The particles are coated with the DNA molecule of interest and accelerated and fired at high speed by the shock wave generated by the helium gas discharge under high pressure. When colliding with a target cell or tissue, it penetrates the cell wall and plasma membrane without destruction (Klein et al. (1987), Nature 327: 70-73), and the endogenous DNA is microscopic by the action of the cell fluid. Must be dissociated from and integrated into the plant genome. After being cultured and regenerated in vitro, these transgenic tissues or cells give rise to genetically modified plants (Brasilio and Aragao (2001), Plant Biotechnol J.3 (3): 113-121; Rech et al. (2008), Nat Protoc 3: 410-418).

Seed decontamination Soybean seeds derived from cultivars BRS133 and Pintado, which are both sensitive to root-knot nematodes, were used. After weighing, the seeds were decontaminated by soaking in 70% ethanol for 10 minutes and then in 1% sodium (V / V) for 20 minutes. In the laminar flow hood, the seeds were washed 3 times with autoclaved distilled water, embedded in twice the amount of water of the seeds and continued to soak for hydration for about 16-18 hours.

Embryo isolation and preparation of support plates for collision Embryos were isolated from hydrated seeds using a dehumidifier and an air conditioner with low room humidity. With the help of sterile forceps and surgical blades, seeds were incised, the hypocotyls were removed and transferred to a petridish containing distilled water to avoid drying. Next, with the help of a stereomicroscope, the leaf primordia were excised to expose the apical meristem region. The embryos are transferred to a filter paper in a laminar flow hood to remove excess water, which can reduce the efficiency of transformation, which acts as a barrier to refracting particles. For collisions, 16 mm diameter central salt (dead zone) was used with sterile forceps in a small petridish containing MS medium (Murashige and Skoog Salt), 3% sucrose and 0.8% fitagel (pH 5.7). Designed. With the help of a stereomicroscope, embryos were placed in grooves in the plate with the apical meristem region facing up. The carrier membrane support and support cylinder were fire sterilized and the four disrupted membrane sets were separated and continued to be immersed in isopropanol until use. Carrier membranes were collected in their supports.

Sterilization and cleaning of tungsten particles In a centrifuge tube, weigh 60 mg of M10 tungsten particles, which is sufficient material for about 100 firings. To this centrifuge tube was added 10 mL of 70% ethanol; the solution was vigorously homogenized and maintained on a stirrer at a rate sufficient to maintain motion for 15 minutes. A 5-minute centrifugation step was performed at 15,000 g and the supernatant was removed and discarded with the help of a micropipette to avoid inhibition of particulate settlement. 1 mL of sterile distilled water was added to the tube, the mixture was vigorously homogenized using a stirrer and again centrifuged at 15,000 g for 5 minutes. The supernatant was discarded and the washing step was repeated twice. After final washing and discarding the supernatant, the microparticles were resuspended in 1 mL of 50% glycerol (V / V).

DNA coating of tungsten microparticles 50 μL of microparticle suspension was dispensed into a 1.5 mL centrifuge tube and subjected to a sonicator for 15 minutes for homozygousization, thus avoiding microparticle aggregation and uniform precipitation. Made possible. A minimum of 6 μg of equivalent DNA was added to the supernatant and gently homogenized with the help of a micropipette. 50 μL of CaCl ₂ (2.5 M) was added to the solution, gently homogenized, and then 20 μL of spermidine was added. After homozygation, the solution was incubated in a suitable tube stirrer for 10 minutes at room temperature with gentle rotation. The tube was allowed to settle at maximum rate for 10 seconds, the supernatant was carefully removed and discarded to avoid resuspension of the particulates. 150 μL of 100% ethanol was added; the solution was vigorously homogenized and centrifuged again at maximum speed (spin) for 10 seconds. The supernatant was removed and the washing step was repeated once more. After discarding the supernatant, in the final wash, 24 μL of 100% ethanol was added to the vigorously homogenized sample. Finally, a 3.2 μL aliquot of the supernatant was applied to the central region of each carrier membrane pre-positioned on the membrane support. Each precipitate is sufficient to prepare six carrier membranes containing DNA-coated microparticles. After application of the microparticles, the membrane was immediately stored on a plate containing silica gel and maintained in the dissection chamber. This is because exposure of DNA-coated microparticles to air relative humidity conditions greater than 50% allows for aggregation, resulting in a reduced frequency of foreign gene expression.

Use of genetic guns and collision experiments With some modifications, the varieties BRS133 and are susceptible to root-knot nematodes according to the protocol developed by Aragao et al. (Crop. Sci. 42: 1298-1302 (2002)). The meristem site of soybean seed embryos derived from Pintado was targeted for launch. Prior to collision, the laminar flow hood was cleaned with 70% ethanol and sterilized by UV irradiation (15 minutes). A retention screen, a petri dish containing embryos, a set of four, a disrupted membrane immersed in isopropanol, and a carrier membrane containing foreign DNA were maintained close to the operator. The valve of the helium gas cylinder was opened and the pressure was set to 1.200 psi. The fracture membrane set (300 psi / membrane) was then placed at the far end of the high pressure helium gas chamber, the sealing screw was tightly closed, the high pressure chamber was inverted and fitted into the vacuum chamber. A plate containing the material to be abutted was placed and a retention screen and carrier membrane support containing DNA-coated microparticles were also placed on the support cylinder. Carefully placed the support cylinder and closed the vacuum chamber. The value allowing vacuum was gently opened in the chamber until the pressure reached 27 pol / Hg, at which time the valve was closed. A valve was opened to push helium gas into the high pressure chamber, and when the fracture membrane showed bending due to the presence of helium gas, it was fired by pushing the trigger. After firing, the valve was closed, while the valve that released helium gas from the high pressure chamber was opened. The vacuum-releasing valve was then slowly opened and the plate containing the embryos to be collided was removed from the device. In each collision, these steps were repeated. After each collision, most embryos were transferred to different plates and subjected to abiotic and biological stress. However, some embryos were transferred to plates containing MS medium supplemented with BAP 5 mg / mL (benzylaminopurine) 3% sucrose, 0.6% agar (pH), in which, for induction of regeneration, It was maintained for 18 hours and protected from light at 28 ° C.

Selection of transgenic embryos To distinguish between transgenic and non-transgenic cells, a selection gene that expresses a protein with enzymatic activity is introduced into the plant genome, either individually or by linking to a gene of interest in the same transformation vector. It can be done by. According to the mode of action, marker genes are classified as genes that confer resistance to antibiotics, genes that confer resistance to herbicides, and marker genes with positive selection (Brasileiro and Dushi (1999), In: TORRES). , AC; CALDAS, L.S .; BUSO, JA Ed. Cultura de tecidos. And transformagao genetica de Plantas. Brasilia: Embrapa-SPI / Embra 2). The gene ahas is classified as a gene that confer resistance to herbicides. This gene encodes a modified form of the acetolactate synthase (AHAS) enzyme and is also known as acetolactate synthase (ALS). Mutation of this sequence to position 653 results in the substitution of serin by asparagine, resulting in a modifying enzyme, which is not recognized by the herbicide class imidazolinone. Meristem cells transformed with this gene are selected in the presence of the herbicide Imazapyr in a process based on the total transfer of selected molecules to the meristem site and inactivation of the endogenous AHAS enzyme. Since the selective agent is localized in the apical meristem, non-transgenic cells are killed and the survival of transgenic cells growing in the plant is supported (Aragao and Brasileiro (2002), JP Physio, 14 (I). )). In this way, after regeneration, some embryos are transferred to a plastic cup containing selective medium MS, 3 & sucrose, 0.15 μM Imazapyr herbicide, 0.8% agar and B5 vitamin (pH 5.7). It was grown in a growth chamber at 28 ° C. for about 45 days with a light cycle of 16 hours. The light intensity was 50 μmol m-2s-1, and the relative humidity exceeded 80%. The regenerated plants were then transferred to a plastic cup containing autoclaved sand: vermiculite (1: 1) and retouched with nutrient solution (pH 6.6). Later, for acclimatization, the cups were covered with a plastic bag, sprinkled with nutrient solution as needed, and grown in a growth chamber for an additional 28-30 days. After this organ of acclimation, the plastic bag can be removed and allowed to grow normally as long as sample collection is possible for molecular analysis and identification of positive plants via PCR techniques with specific primers. rice field.

Experiments with transgenic embryos subjected to various stresses Sensitive to root-knot nematodes and various expression cassettes (parent-sensitive strain BRS133 (S-pAG1 / promoter GmHSP) Embryos derived from soybean varieties BRS133 and Pintado transformed with BRS133) and individuals derived from the resistant population JF7027 (including the promoter region of the Gmhsp17.6-L gene amplified from R-pAG1 / promoter GmHSP JF7027) are living organisms. Stress, in this case M. Javanica was inoculated with Javanica J2 and subjected to abiotic stresses such as heat, cooling, salt and dehydration (drought). Non-transgenic embryos (CN-negative subjects) and embryos transformed with only the pAG1 plasmid (CP-positive subjects) were subjected to the same treatment. Heat stress was applied and embryos were maintained at 25 ° C. (room temperature), 35 ° C. and 45 ° C. (in a greenhouse). Treatments at 4 ° C (refrigerator) and 15 ° C (greenhouse) were used for cooling stress. These stresses were applied to the embryos in solution at 2, 4, and 24 hours. Dehydration (drought) was performed in a greenhouse at 37 ° C. and maintained on filter paper at 2 hours, 4 hours, and 6 hours. For salt stress, embryos were maintained at NaCl concentrations of 200 mM and 400 mM for 24 hours. Biological stress was performed by maintaining the embryos in a solution containing nematodes at the J2 developmental stage (2.00-3.000 J2 / mL) for 24 hours, 48 hours, and 72 hours. FIG. 17 shows how stress was applied.

Histochemical assay in embryos transformed with various expression cassettes and subjected to various stresses The expression product of the Gus gene is detected by detecting its enzymatic activity in plant tissue through histochemical assays. Can be used as a marker reporter of choice. This quantitative method uses the b-glucuronidase enzyme to produce a dimer that results in an insoluble blue precipitate in the presence of oxygen, 5-bromo-4-chloro-3-indrill-b-D-glucuronide (X). -Gluc) Based on cleavage of the substrate. This methodology assists in gene expression control studies on tissue specificity, promoter isolation, transgenic plant identification, and migration conditions (Brassileiro (1998), In: Brasileiro, A.C.M .; Carneiro, V. TC (ed.). 101: 1-6)). In this way, embryos of the two genotypes BRS133 and Pintado, transformed in different cassettes and subsequently subjected to different stresses, were transferred to a plate in a volume sufficient to cover the sample. Gluc reaction buffer was added. This buffer was prepared by diluting 8.5 mg of X-Gluc in 85 mL of dimethylformamide. Phosphate buffer (NaH2PO4-50 mM pH 7.0) was added to a volume of 17 mL, and finally 17 mL of Triton X-100. After preparing this solution, it was stored in the refrigerator. Plates containing embryos and buffer were sealed and incubated in the dark at 37 ° C. for about 16 hours. The buffer was then removed and 1 mL of 70% ethanol was added to terminate the reaction. The sample was analyzed under a stereomicroscope (SQZ-DS4-BI (Technival)), images were acquired and the results were documented. Histochemical assays showed that cultivar BRS133 showed a stronger response when subjected to reagent X-Gluc after stressing, which showed clear blue spots and transformed cells. This is because it is visible in the restricted area shown. This insoluble blue precipitate is a Gus gene reaction product present in the construct cassette encoding β-glucuronidase, which reacts with the X-Gluc substrate, which forms a dimer that precipitates in the presence of O2. .. When analyzing embryonic assay results from cultivar Pintado, no positive blue spots were detected for most of the stress. In this variety, endogenous GUS expression is higher, resulting in a complete blue embryo, the result of which interferes with the analysis. Background endogenous GUS activity in soybean embryos has been reported in previous studies. Hu and collaborators (Plant Cell Reports, 9: 1-5, 1990) analyzed 53 different plant species and tested their unique GUS activity in leaves, fruit parts, seeds and embryos. In this study, soybeans showed strong positive staining in mature embryos derived from fresh fruits and mature embryos of dehydrated seeds, demonstrating the results of endogenous GUS activity. In this way, heat stress was applied by subjecting transgenic embryos to temperatures of 25 ° C, 35 ° C, and 45 ° C for 2 hours, 4 hours, and 24 hours in a greenhouse. Figures 18, 19 and 20 show these treatments for both varieties tested, respectively. Cooling stress was applied by subjecting transgenic embryos to temperatures of 4 ° C and 15 ° C for 2 hours, 4 hours, and 24 hours in a refrigerator and greenhouse (FIGS. 21 and 22). For this stress, the differences between cultivars with respect to the positive blue spots of histochemical reactions are more pronounced in Pintado embryos, they are structurally blue and are due to endogenous GUS activity. It is described in the previous literature (Hu et al. (1990), Plant Cell Reports, 9: 1-5). Once again, embryos from cultivar BRS133 show a better response to the histochemical assay, indicating that this cultivar is the one of choice and must be used in the next step of this study. .. Salt stress experiments were performed by maintaining transgenic embryos in water as a control, NaCl concentrations of 200 mM, 400 mM for 24 hours. Embryos from cultivar BRS133 continued to respond positively to this assay, showing blue spots in restricted areas, and unlike previous stress, cultivar Pintado did not show a clear and detectable response. FIG. 23 shows those results. Drought stress was applied by placing embryos on filter paper at 37 ° C. and maintaining in a greenhouse for 2, 4, and 6 hours. Once again, cultivar BRS133 gave a positive response to the histochemical assay, while cultivar Pintado again gave a negative response. The latter showed non-specific blue staining of embryos as a result of endogenous GUS expression, as shown in FIG. Root hump nematode M. Using Javanica J2, which is the infection stage of Javanica, infection of embryos from cultivars BRS133 and Pintado established biological stress. Approximately 2000-3000 J2 / mL was incubated with transgenic samples and maintained for 24 hours, 48 hours and 72 hours. The results indicate that cultivar BRS133 (FIG. 25) showed a positive blue spot in the histochemical assay, indicating that cassette expression occurred satisfactorily. After this time, the embryos have their aspects modified and show a reddish-brown color, indicating that they contain a control sample. This change is more visible after 72 hours and can be caused by attacking the embryo by the infection form of the stain, which injects hormones and substances at the time of infection, as it occurs in the roots of the host plant. Allows cell modification. However, the embryo named R is a resistant cassette pAG1 / promoter Gmhsp. Samples transformed with JF7027 and having a less affected aspect, samples inoculated at 72 hours maintained the original aspect and were white, which apparently resulted in a higher number of ATs ( n) Repeatedly, Gmhsp17. From individuals from the resistant population JF7027. It is suggested that cassettes containing the promoter region of the β-L gene show a better response to pathogen attack, probably with higher expression levels of chaperones. Pintado-derived embryos also responded to color change in addition to a negative response to a histochemical assay that was constitutively stained blue when subjected to biological stress by inoculation with J2 nematodes. It was shown and was more pronounced from 48 hours, especially from 72 hours. FIG. 26 shows the results.

After being subjected to biological and abiotic stress, histochemical assays detected the presence of the marker gene Gus, which can be concluded from the results shown for varieties BRS133 and Pintado, but with high endogeneity. Sexual activity was also observed, especially in the cultivar Pintado, suggesting that different materials showed a clear response to the transformation process.

It is worth mentioning that Gus expression is just considered as evidence of transformation, but insufficient as conclusive evidence of integration of the Gus gene into the plant genome (Potrykus (1990), Physiologia Plantarum 79: 129). -134). Conclusive evidence of integration of endogenous DNA sequences is provided by genomic DNA hybridization using specific probes via molecular biology techniques.

Claims (5)

A method for upwardly controlling the expression level of a target gene in a soybean plant.
(I) Stable transformation of soybean plant cells using an expression cassette containing a promoter element operably linked to a target gene, wherein the promoter element is 9 to 9 of the "AT" nucleotide sequence. It contains an insert consisting of 32 iterations, wherein the promoter element is:
gaattctgaa attgggtctt tttgtgggca ctttttgatg tttttgttta agttactgta ctgtgggcca caaaacgtat agatcaaagt agtaataata atattgatta aatgatatat atatatatat atatatatat atatatatat atatatatat atatatatat atatatctag aaggttgtag aagactagct agaacgtacg tattcgtgtg gagaagtcct gaagtttatc gaatcatcta aaactgctaa aatagcaaac aacattatat tgtaaacaat atttttctgg aacatacaag agtatccttt cacttccttt aaatacctcg agtgtcccca ttgac;
gaattctgaa attgggtctt tttgtgggca ctttttgatg tttttgttta agttactgta ctgtgggcca caaaacgtat agatcaaagt agtaataata atattgatta aatgatatat atatatatat atatatatat atatatatat atatatatat atatatatat atatatatct agaaggttgt agaagactag ctagaacgta cgtattcgtg tggagaagtc ctgaagttta tcgaatcatc taaaactgct aaaatagcaa acaacattat attgtaaaca atatttttct ggaacataca agagtatcct ttcacttcct ttaaatacct cgagtgtccc cattgac;
gaattctgaa attgggtctt tttgtgggca ctttctgatg tttttgctta agttactgta ctgtgggcca caaaacgtat agatcaaagt agtaataata atattgatta aatgatatat atatatatat atatatatat atatatatat atatatatat atatatatat atatatctag aaggttgtag aagactagct agaacgtacg tattcgtgtg gagaagtcct gaagtttatc gaatcatcta aaactgctaa aatagcaaac aacattatat tgtaaacaat atttttctgg aacatacaag agtatccttt cacttccttt aaatacctcg agtgtcccca ttgac; or,
gaattctgaa attgggtctt tttgtgggca ctttttgatg tttttgttta agttactgta ctgtgggcca caaaacgtat agatcaaagt agtaataata atattgatta aatgatatat atatatatat atatatatat atatatatat atatatatat atatatatat atatctagaa ggttgtagaa gactagctag aacgtacgta ttcgtgtgga gaagtcctga agtttatcga atcatctaaa actgctaaaa tagcaaacaa cattatattg taaacaatat ttttctggaa catacaagag tatcctttca cttcctttaa atacctcgag tgtccccatt gac
Is;
(Ii) Stable transformed soybean plant cells are cultured under the growth conditions of soybean plant cells;
(Iii) cassette (i) is regenerating a transgenic soybean plant stably integrated into its genome; and (iv) the transgenic soybean plant, and this applying the nematodes stress,
Wherein the wherein the transgenic soybean plants, compared with the case of not applying the nematodes stress, the method shown Target gene higher gene expression levels. Nematodes used in the prior SL nematodes stress is a root-knot (gall) nematodes Meroidogine-Jawanika (Meloidogyne javanica), The method of claim 1. An expression cassette, a soybean plant transformed by applying the nematodes stress soybean plants transformed with said expression cassette, an expression cassette for upregulating the expression level of the gene of interest, It comprises a promoter element operably linked to a gene of interest, wherein the promoter element has an insert consisting of 9 to 32 repetitions of the "AT" nucleotide sequence, wherein the promoter element comprises.
gaattctgaa attgggtctt tttgtgggca ctttttgatg tttttgttta agttactgta ctgtgggcca caaaacgtat agatcaaagt agtaataata atattgatta aatgatatat atatatatat atatatatat atatatatat atatatatat atatatatat atatatctag aaggttgtag aagactagct agaacgtacg tattcgtgtg gagaagtcct gaagtttatc gaatcatcta aaactgctaa aatagcaaac aacattatat tgtaaacaat atttttctgg aacatacaag agtatccttt cacttccttt aaatacctcg agtgtcccca ttgac;
gaattctgaa attgggtctt tttgtgggca ctttttgatg tttttgttta agttactgta ctgtgggcca caaaacgtat agatcaaagt agtaataata atattgatta aatgatatat atatatatat atatatatat atatatatat atatatatat atatatatat atatatatct agaaggttgt agaagactag ctagaacgta cgtattcgtg tggagaagtc ctgaagttta tcgaatcatc taaaactgct aaaatagcaa acaacattat attgtaaaca atatttttct ggaacataca agagtatcct ttcacttcct ttaaatacct cgagtgtccc cattgac;
gaattctgaa attgggtctt tttgtgggca ctttctgatg tttttgctta agttactgta ctgtgggcca caaaacgtat agatcaaagt agtaataata atattgatta aatgatatat atatatatat atatatatat atatatatat atatatatat atatatatat atatatctag aaggttgtag aagactagct agaacgtacg tattcgtgtg gagaagtcct gaagtttatc gaatcatcta aaactgctaa aatagcaaac aacattatat tgtaaacaat atttttctgg aacatacaag agtatccttt cacttccttt aaatacctcg agtgtcccca ttgac; or,
gaattctgaa attgggtctt tttgtgggca ctttttgatg tttttgttta agttactgta ctgtgggcca caaaacgtat agatcaaagt agtaataata atattgatta aatgatatat atatatatat atatatatat atatatatat atatatatat atatatatat atatctagaa ggttgtagaa gactagctag aacgtacgta ttcgtgtgga gaagtcctga agtttatcga atcatctaaa actgctaaaa tagcaaacaa cattatattg taaacaatat ttttctggaa catacaagag tatcctttca cttcctttaa atacctcgag tgtccccatt gac
The expression cassette. The expression cassette according to claim 3, wherein the nematode is a root-knot nematode, Meloid Gine Javanica. A method for obtaining a gene-modified soybean plant,
Before Symbol method comprises the steps of:
(I) Stable transformation of soybean plant cells using an expression cassette containing a promoter element operably linked to a target gene, wherein the promoter element is 9 to 9 of the "AT" nucleotide sequence. It contains an insert consisting of 32 iterations, wherein the promoter element is:
gaattctgaa attgggtctt tttgtgggca ctttttgatg tttttgttta agttactgta ctgtgggcca caaaacgtat agatcaaagt agtaataata atattgatta aatgatatat atatatatat atatatatat atatatatat atatatatat atatatatat atatatctag aaggttgtag aagactagct agaacgtacg tattcgtgtg gagaagtcct gaagtttatc gaatcatcta aaactgctaa aatagcaaac aacattatat tgtaaacaat atttttctgg aacatacaag agtatccttt cacttccttt aaatacctcg agtgtcccca ttgac;
gaattctgaa attgggtctt tttgtgggca ctttttgatg tttttgttta agttactgta ctgtgggcca caaaacgtat agatcaaagt agtaataata atattgatta aatgatatat atatatatat atatatatat atatatatat atatatatat atatatatat atatatatct agaaggttgt agaagactag ctagaacgta cgtattcgtg tggagaagtc ctgaagttta tcgaatcatc taaaactgct aaaatagcaa acaacattat attgtaaaca atatttttct ggaacataca agagtatcct ttcacttcct ttaaatacct cgagtgtccc cattgac;
gaattctgaa attgggtctt tttgtgggca ctttctgatg tttttgctta agttactgta ctgtgggcca caaaacgtat agatcaaagt agtaataata atattgatta aatgatatat atatatatat atatatatat atatatatat atatatatat atatatatat atatatctag aaggttgtag aagactagct agaacgtacg tattcgtgtg gagaagtcct gaagtttatc gaatcatcta aaactgctaa aatagcaaac aacattatat tgtaaacaat atttttctgg aacatacaag agtatccttt cacttccttt aaatacctcg agtgtcccca ttgac; or,
gaattctgaa attgggtctt tttgtgggca ctttttgatg tttttgttta agttactgta ctgtgggcca caaaacgtat agatcaaagt agtaataata atattgatta aatgatatat atatatatat atatatatat atatatatat atatatatat atatatatat atatctagaa ggttgtagaa gactagctag aacgtacgta ttcgtgtgga gaagtcctga agtttatcga atcatctaaa actgctaaaa tagcaaacaa cattatattg taaacaatat ttttctggaa catacaagag tatcctttca cttcctttaa atacctcgag tgtccccatt gac
Is;
See contains at reproducing and (iii) stably genetically modified soybean plant,; (ii) stable under growth conditions of the soybean plant cell and culturing the transformed soybean plant cells
The method, wherein the genetically modified soybean plant overexpresses the target gene by applying nematode stress .

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