JP2500321B2 - Novel DNA strand - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a cloned DN of allyl acylamidase.
A, fragments thereof, and plasmids incorporating the cloned DNA or fragments described above.

[Conventional technology]

Propanil (3,4-dichloropropionanilide)
Is a herbicide used for rice. Weeds die but rice is resistant. This is because rice has the enzyme allyl acylamidase that specifically decomposes and detoxifies propanil (Science, 160, 1360, 1968).
Allyl acylamidase that decomposes propanil is not only rice, but also parsley and tulip (Phytochemistry, 11, 5
21-527, 1972), etc., and is also contained in very limited plants.

However, allyl acylamidases of higher plants have not been completely purified, and there are few findings on this enzyme. Moreover, the cloning of the allyl acylamidase gene has not been performed at all.

On the other hand, in the genetic manipulation of plants, in order to select only cells or plants in which the gene of interest has been incorporated, it is necessary to introduce a drug resistance selection gene by linking it to the gene to be introduced into the plant. It is common. The kanamycin resistance gene is most often used as a drug resistance gene for that purpose. However, since the kanamycin and kanamycin resistance genes were not originally developed for genetic engineering of plants, their performance is not very high, and selection may not be successful depending on the plant species. Hygromycin resistance gene etc. are also used to compensate for these drawbacks, but in any case, it is a microorganism-derived antibiotic resistance gene,
These are considered to be unsuitable for genetic engineering of practical plants, especially food crops, due to environmental problems and safety problems (plant cell engineering, 1,62, 1989).

Further, as for the range of agricultural use of the herbicide propanil itself, most of the crops do not have allyl acylamidase activity, so that they are mainly used for rice and the crops that can be used are limited.

[Problems to be Solved by the Invention]

As a selectable marker gene required for genetic engineering of plants, it is considered that genes cloned from plants or the like having less safety anxiety are more desirable than microbial genes. Currently, the neomycin phosphotransferase II gene (NPT II) and the hygromycin phosphotransferase gene, which are mainly used at present, have the drawbacks described in the above "Prior Art". Furthermore, it is considered to be undesirable in view of the fact that the drug resistant by these genes is an antibiotic itself, in particular, its application to food crops, and the problem of environmental release. If we could clone the higher plant allyl acyl amidase gene, we would not have these drawbacks,
Furthermore, it is expected to become a selectable marker gene that meets the needs for practical application. Furthermore, since the allyl acylamidase gene is originally a herbicide resistance gene, it can exert a higher selection pressure than conventional selection markers, and is expected to have an advantage as a further excellent selection marker gene for genetic engineering.

On the other hand, in addition to the use as a marker gene, the allyl acylamidase gene imparts propanyl resistance to plants and the like by being introduced into plants and the like, and can be used to prepare herbicide-resistant plants and the like. Be expected.

[Means for solving the problem]

The present inventor has found that the allyl acylamidase of tulip is optimal as a result of earnest studies for solving the above-mentioned problems, and arrived at the present invention.

That is, the first aspect of the present invention relates to a DNA chain having the base sequence shown in FIG. 1 which encodes the amino acid sequence of tulip allyl acylamidase. This DNA chain is the tulip allyl acylamidase gene.

A second aspect of the present invention relates to a plasmid containing the DNA strand.

The DNA chain of the present invention can be obtained as follows.

First, allyl acylamidase was purified from tulip and injected into Uzaki to obtain antiserum. Next, the amino acid sequence was partially determined by analyzing the protein of allyl acylamidase. Next, the allyl acylamidase gene (NDA chain) was cloned from tulip using the obtained antiserum as a probe. As a result of structural analysis of the obtained gene (DNA chain), a sequence encoding the amino acid sequence obtained by protein analysis is contained,
This confirmed that the obtained gene (DNA chain) was an allyl acylamidase gene.

The amino acid sequence of the tulip allyl acylamidase of the present invention is shown in FIG. 3, and the nucleotide sequence encoding the amino acid sequence of the tulip allyl acylamidase of the present invention is shown in FIG.

For the phage or plasmid containing the second tulip allyl acylamidase gene of the present invention, a c-DNA library is prepared from tulip m-RNA or a genomic library is prepared from nuclear DNA according to a known conventional method. It can be isolated by preparing it and screening it by the immuno detection method using a tulip allyl acylamidase antiserum. Alternatively, it can be isolated by screening from the above-mentioned c-DNA library or genomic library by using an oligonucleotide synthesized from the nucleotide sequence shown in FIG. 1 as a probe and by a known conventional method. . In addition, the tulip allyl acylamidase gene has the following nucleotide sequence shown in FIG.
Using the synthesized oligonucleotide as a primer, according to a known conventional method, from the nuclear DNA of tulip or the tulip c-DNA, the polymerization chain
Reaction method (abbreviated as PCR method: Science.230,1350)
-1354, 1985) can be directly isolated.

The plant, animal or microorganism containing the DNA chain can be produced as follows.

The tulip allyl acylamidase gene of the present invention is used as an expression vector for a suitable plant, for example, PBI121 (manufactured by Clontech), BIN19 (Nucleic, Acid, Research, 12,87).
11-8721, 1982), etc., to construct an expression vector for producing tulip / allyl acylamidase in a plant or the like. In this way, the expression vector constructed by linking the allyl acylamidase gene, for example, by a plant transformation method with Agrobacterium, for example, introduced into tobacco, potato, arabidopsis, asparagus, tomato, etc., Propanyl resistance can be given to plants,
Can produce herbicide-resistant crops. In addition, suitable promoters, such as cauliflower mosaic virus 35S-promoter, nopaline synthetase promoter and the like linked to tulip allyl acylamidase, for example, electroboration method, for example, by particle gun method, for example, soybean, It can be introduced into plants such as rice and corn, animals or microorganisms to impart propanil resistance to the plants. Furthermore, a gene to be introduced into a plant or the like is ligated to a tulip / allyl acylamidase, and when the gene is introduced into the plant or the like using the same method as described above, the allyl acylamidase gene expressed in the plant or the like is introduced. Can be used as a drug selection marker gene to select only transformed plants.

〔The invention's effect〕

Since the DNA chain of the present invention has the activity of degrading propanil which is a herbicide in a host, especially a plant, etc., it is effectively used as a drug selection marker in gene recombination technology or for the production of herbicide-resistant plants etc. be able to.

[Examples, etc.]

Production Example 1 Cloning of allyl acylamidase gene Step 1 Purification of tulip allyl acylamidase To a commercially available tulip bulb (variety name: rose beauty), 30 mM phosphate buffer PH7.0 was added and ground with a polytron homogenizer. 10,000 in a fast cooling centrifuge
Cell debris was removed by centrifugation at spin for 10 minutes. Ammonium sulfate was added to the supernatant to a saturated concentration of 40%, and the precipitate was removed by centrifugation at 10,000 rpm for 10 minutes. Ammonium sulfate was added to the supernatant so that the concentration became 60%, and the precipitate was collected by centrifugation at 10,000 rpm for 10 minutes.

The crude protein suspension thus obtained was purified in the next four steps.

1. Removal of low molecular weight substances by dialysis.

2. Diethylaminoethyl cellulose chromatography 3. Gel filtration chromatography with Toyopearl HW55F 4. Hydroxyl apatite chromatography The purified protein is Laemmli's method (Nature, 227.6).
80, 1970) and confirmed to be pure by SDS polyacrylamide gel electrophoresis and high performance liquid chromatography.

Step 2 Determination of amino acid partial sequence.

Lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.), which is a proteolytic enzyme, was added to the purified allyl acylamidase protein to perform limited decomposition. After that, the protein fragments are separated by SDS polyacrylamide gel electrophoresis,
This was electrophoretically transferred to a synthetic resin film, Immobilon (manufactured by Millipore). Using this as a sample, ABI model 477
The amino acid sequence was determined by Edman degradation using A gas phase protein sequencer.

Step 3 Preparation of antiserum Rabbit anti-allyl acylamidase serum against purified allyl acylamidase was prepared by the following method.
1.0 mg / ml concentration of allyl acylamidase solution (30 mM,
A suspension of 1.0 ml of pH 7, phosphate buffer / solution) in an equal amount of Freund's complete adjuvant (manufactured by Difco) was injected into a rabbit as an antigen, and 4 weeks later, the same operation was performed. Two weeks later, 2 ml of blood was collected. Then, the obtained blood was allowed to stand in a refrigerator for 2 hours, and this was centrifuged at 7,000 rpm for 15 minutes with a centrifuge to obtain anti-allyl acylamidase serum as a supernatant. This antiserum had a titer sufficient to detect allyl acylamidase spotted on the nitrocellulose membrane at a concentration of 100 pg / mm.

Step 4 Cloning of tulip allyl acylamidase (a) Preparation of poly (A) RNA containing m-RNA Total RNA was prepared from the tulip bulb using the SDS / phenol method. 90 to 10g of tulip bulbs
% Phenol solution (40 ml) and 1% SDS solution (40 ml) were added, and the mixture was ground with a blender. After centrifugation at 10,000 rpm for 10 minutes, the supernatant was taken, 20 ml of 90% phenol and 20 ml of chloroform were added and mixed, and the mixture was centrifuged at 10,000 rpm for 10 minutes. 10 in the supernatant
Add 0.5 ml of lithium chloride M and 10 ml of ethanol, and then -20 ℃
Then, the mixture was left standing for 1 hour, centrifuged at 10,000 rpm for 10 minutes, and then 5 ml of water was added to the precipitate to dissolve it. 5 ml of 10M lithium chloride was added, and the mixture was left at -20 ° C for 1 hour, centrifuged at 10,000 rpm for 10 minutes. Repeat this operation again, then 70
It was washed 3 times with% ethanol to obtain a total RNA fraction.

From this total RNA, oligos were prepared according to the method of Aviv and Leder.
poly by dT cellulose column chromatography
(A) RNA fraction was purified (Proceeding of Nationa
l Academy of Science USA 69, 1408, 1972). That is, 10 mM Tris-HCl buffer solution containing 0.5 M KCl (PH7.
Poly (A) RNA was obtained by binding to oligo dT cellulose using 5) as a binding buffer and then eluting with a buffer containing no KCl.

(B) Preparation of c-DNA library From poly (A) RNA, cDNA synthesis was performed using Gudler and hoffman.
Based on the method of n (Gene, 25, 263-269, 1983), the "cDNA synthes" manufactured by Pharmacia, which has been improved.
is kit ”.

The operation was performed according to the manual attached to this kit. The outline of the operation is shown. Poly obtained from tulips
(A) Add water to 5 μg of RNA to make 20 μm. This sample is heated at 65 ° C for 10 minutes and then cooled to 0 ° C.
First-strand synthesis reagent (reverse transcriptase, oligo dT primer, dN
The main component is TPs. ) Is added and reacted at 37 ° C. for 1 hour. Second-strand synthesis reagent (RNaseH, DNA polymerasel, dNTPs as main components) is added, and the mixture is reacted at 12 ° C for 1 hour and then at 22 ° C for 1 hour. Add 1 unit of Klenow fragment of DNA polymerase and incubate at 37 ° C for 30 minutes. Add 50μ of chloroform and 50μ of phenol and stir.
The reaction solution is centrifuged at 15,000 rpm for 1 minute with a microcentrifuge, and the supernatant is applied to a Sephacryl S200 gel filtration column. Add 5 µl of EcoRl adapter and 1 µl of ATP solution 3 µl of T4 DNA ligase to the eluate and incubate at 12 ° C overnight. Heat at 65 ° C for 10 minutes and cool to 0 ° C. 10μ ATP solution and 1μ
Add T4 polynucleotide kinase from and add and incubate at 37 ℃ for 30 minutes. Add 50μ phenol and 50μ chloroform and stir. The reaction solution is centrifuged at 15,000 rpm for 1 minute with a microcentrifuge, and the supernatant is applied to a Sephacryl S200 gel filtration column. The eluate is the synthesized cDNA.

By performing the above operation, cDNA was synthesized using 5 μg of tulip poly (A) RNA. The cDNA thus obtained was ligated to EcoRl and alkaline phosphatase-treated λ-gt11.phage DNA (manufactured by Stratagene). The ligation of c-DNA and λ-gt11.DNA was ligated using a DNA ligation kit (Takara Shuzo) based on the ligation reaction of DNA by T4, fuzzy, DNA ligase. The reaction procedure is “Nucleic”
Acidsresearch, 14, 7617-7631, 1986 ”.

One-fifth of the thus obtained c-DNA-ligated λ-gt11 was used for packaging into phage particles.
This operation was carried out by packaging the phage particles using "Guide Pack Plus", a kit manufactured by Stratagene. In this way, the λ-gtll phage in which the cDNA was incorporated was reconstructed and used as a cDNA library. This DNA library contained 1 million clones.

(C) Cloning The cloning of the allyl acylamidase gene from the c-DNA library of λ-gt11 was performed by the usual immunoscreening method (Young, Davis, Proc.
eeding of National Academy of Science USA 80,1194
-1198, 1983). That is, this library was spread together with the host Escherichia coli Y1090 on 10 plates so that 5,000 clones per plate, phage plaque formation, nitrocellulose membrane (nitroplus 200
0; manufactured by Micron Separation Co.). This nitrocellulose membrane was treated with a TBSM solution (30 mM Tris-hydrochloric acid buffer pH 8.0 containing 2% skim milk and 100 mM NaCl) and then treated with a rabbit allyl acylamidase antiserum diluted 1,000 times with the TBSM solution for 30 minutes. Next, the nitrocellulose membrane was washed and treated with a peroxidase-conjugated secondary antibody (manufactured by Bio-Rad Laboratory) diluted 3,000 times with TBSM solution for 30 minutes. Using Konica Immunostain (manufactured by Konica) as a coloring reagent, the protein produced by the expression vector λ-gt11 phage was screened with antiserum to obtain λ-gt11 phage having the target gene. . About 10 obtained by (b)
About 50,000 phages out of 0,000 phages were screened using a rabbit allyl acylamidase antiserum as a probe, and about 18 positive clones were obtained. Twelve of these positive clones were cleaved with restriction enzyme EcoRI (TOYOBO), and the inserted c-DNA was analyzed by agarose gel electrophoresis.
The inserted c- of about 1.3 kb to 1.4 kb with about the same length
Since it contained DNA, it was further digested / analyzed with the restriction enzyme HaeIII (manufactured by TOYOBO) about these nine, and as a result, they showed essentially the Onoji restriction enzyme cleavage pattern. The nucleotide sequence of the clone was determined.

(D) Nucleotide sequence determination of cloned allyl acylamidase and corresponding amino acid sequence As a nucleotide sequence determination method for cloned allyl acylamidase, M13 phage and fluorescent primer A were used.
It was performed using a kit manufactured by BI and a DNA sequencer manufactured by ABI. Production Example 1 Step 4 (c) for determination of nucleotide sequence
The allyl acylamidase c-DNA obtained by the above was inserted into and ligated to the phage M13mp18 (manufactured by Nippon Gene) and propagated in E. coli JM103 (manufactured by Nippon Gene). The method of kilosequencing was used to create a defective mutant for determining the entire base sequence. Using the deletion kit for kilosequencing (Takara Shuzo Co., Ltd.) and following the Henikoff method (GENE, 28,351-359, 1984), an allyl acylamidase c-DNA fragment was partially deleted from the end D
A group of NAs was prepared, and the nucleotide sequence of these was determined by ABI DNA sequencer. The entire base sequence was determined by linking these sequences.

FIG. 1 shows the entire nucleotide sequence of tulip allyl acylamidase c-DNA. FIG. 2 shows the sequence strategy used for the nucleotide sequence determination of the tulip allyl acylamidase gene.

FIG. 3 shows the amino acid sequence thus determined, which corresponds to the DNA base sequence of allyl acylamidase.

Production Example 2 Production of a plasmid incorporating an allyl acylamidase gene Step 1 Preparation of Ti vector fragment Plasmid PBI121 (manufactured by TOYOBO) was digested with restriction enzymes Xbal and Ec.
It was digested with oRI (manufactured by TOYOBO) and the 11 kb fragment was electrophoretically separated and recovered using low melting point agarose (manufactured by Nacalai Tesque).

Step 2 Preparation of allyl acylamidase c-DNA fragment The following procedure was performed to remove about 60 residues at the 5'end of allyl acylamidase DNA.

Obtained by the procedure of Production Example 1-Step 4 (c) 1.
4kb of allyl acylamidase c-DNA was added to plasmid PU
It was inserted into c18 (Nippon Gene) and propagated in Escherichia coli GM33 which is a dam-minus bacterium. The plasmid obtained by the above operation was named PUc18 / aaa.

(B) Puc18 / aaa restriction enzymes EcoRI and Bcl1 (TOYOBO)
The fragment was cleaved with a 0.3 kb fragment, and the 0.3 kb fragment was electrophoretically separated and collected using low-melting point agarose (manufactured by Nacalai Techs). Furthermore, the 0.3 kb fragment was cleaved with the restriction enzyme Mni1, and the 0.17 kb fragment was electrophoretically separated and recovered from the low melting point agarose (manufactured by Nacalai Tesque).

(B) PUc18 / aaa restriction enzymes Bcl1 and SmaI (manufactured by TOYOBO)
Cleavage was carried out with and the 3.8 kb fragment was electrophoretically separated and recovered using low melting point agarose (manufactured by Nacalai Techs).

(C) Join the two fragments (a) and (b) above,
Proliferated in E. coli HB101. The plasmid obtained by the above operation was named PUc / aaa-de160. PUc / aaa-d
E160 was cleaved with restriction enzymes Xbal and EcoR1, and a DNA fragment in which about 60 residues of the 5'end of allyl acylamidase DNA had been removed was electrophoretically separated and recovered with low melting point agarose (manufactured by Nacalai Tesque, Inc.), and aaa-de160 I named it.

Step 3 Integration of allyl acylamidase gene into Ti vector.

Ligating the PBI121 fragment and the allyl acylamidase gene aaa-de160 obtained in step 1 and step 2,
The plasmid PKAAA was obtained.

This plasmid is designed to express allyl acylamidase activity in plants. Therefore,
Propanyl resistance can be imparted to the introduced plant (4th
See figure).

[Brief description of drawings]

Figure 1 shows the cDNA of tulip allyl acylamidase.
The entire base sequence of is shown. The portion encoding the enzyme protein of allyl acylamidase begins at the ATG (start codon) starting from the 64th position in the base sequence and ends at the stop codon T
It has a coding region of 1047 bases ending in GA and encodes 349 amino acids. FIG. 2 shows a restriction enzyme map of tulip allyl acylamidase cDNA. White arrows indicate the protein coding regions of allyl acylamidase. The black arrow represents the range of the sequence strategy at the time of sequencing, in which the range of the arrow is sequenced at one time. Numbers are diagrams showing the number of base residues in the nucleic acid from the 5'end. FIG. 3 shows an amino acid sequence corresponding to the nucleotide sequence of tulip allyl acylamidase cDNA, and FIG. 4 shows a physical map of plasmid PKAAA. AAA: Allyl acylamidase, gene CaMV35SP, cauliflower mosaic virus 35S promoter, DNA NPTII, neomycin phosphotransferase 2, gene RB, Ti plasmid right border region, DNA LB, Ti plasmid left border region, DNA

Claims (3)

(57) [Claims] 1. A DNA chain having a base sequence encoding the amino acid sequence of tulip allyl acylamidase shown below. 2. The base sequence is as follows:
The described DNA strand. 3. A plasmid containing the DNA chain according to claim 1.