JP2876511B2 - A new gene from parsley - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a cloned DNA of an arylacylamidase derived from parsley, a fragment thereof, and a vector incorporating the cloned DNA or the fragment.

[0002]

2. Description of the Related Art Dichloropropionanilide, especially 3,4-dichloropropionanilide (hereinafter referred to as DC)
PA is sometimes abbreviated as a herbicide used for rice, which causes weeds to die, but rice shows tolerance. This is because rice has an arylacylamidase which is an enzyme that specifically degrades and detoxifies DCPA (Science, 160, 1360, 1).
968). It is known that arylacylamidase that degrades DCPA is contained not only in rice but also in very limited plants such as parsley and tulip (Ph).
ytochemistry, 11, 521-527, 1
972). Although the arylacylamidases of these plants have in common that they degrade a group of compounds belonging to arylacyl, the rice arylacylamidase is different from the arylacylamidase 1 due to the remarkable difference in other enzymatic properties. The type and the acyl acylamidase of parsley are respectively classified into the type 3 aryl acylamidase. Tulip aryl acylamidase is unclassified (Nakami Akatsuka, Weed Research 24)
Vol. 55 to 63, 1979).

[0003] However, research on arylacylamidases of higher plants has not been advanced much, and the complete purification of the enzyme is disclosed in Japanese Unexamined Patent Publication No. 3-290190.
Only the enzyme purification of the tulip according to the publication. As for the cloning of the arylacylamidase gene, only the cloning of the arylacylamidase gene of tulip described in the above-mentioned publication of the present inventors is known.

On the other hand, in genetic engineering of plants, it is necessary to link a gene for drug resistance selection to the gene to be introduced and to introduce it into the plant in order to select only cells or plants into which the target gene has been incorporated. General. As a drug resistance gene for that purpose, a kanamycin resistance gene is most often used. However, kanamycin and the kanamycin resistance gene were not originally developed for genetic engineering of plants, so their performance is not very high, and selection may not be successful depending on plant species. Hygromycin resistance gene and the like are also used to make up for these drawbacks, but in any case, they are antibiotic resistance genes derived from microorganisms, and these are environmentally and safety-related problems, and practical plants, Whether it is particularly suitable for genetic engineering of food crops is controversial (plant cell engineering, 1,62,
1989).

[0005] Furthermore, the agricultural application range of the herbicide DCPA itself is that most crops do not have arylacylamidase activity, and thus are mainly used for rice having arylacylamidase activity. Available crops are limited. If the arylacylamidase gene can be incorporated into various plants,
The range of use of the herbicide DCPA is expected to expand.

[0006]

As a selectable marker gene required for genetic engineering of plants, it is considered that a gene cloned from a plant or the like with less concern about safety is more preferable than a gene of a microorganism. Current,
The mainly used neomycin phosphotransferase II gene (NPTII) and hygromycin phosphotransferase gene have the drawbacks as described in the above-mentioned "prior art". Furthermore, it is a question of whether or not the drug itself resistant by these genes is an antibiotic itself, especially if it is generally accepted in view of the application to food crops and the issue of environmental release. It is. If the arylacylamidase gene of a higher plant can be cloned, it is expected to be a selection marker gene that does not have these disadvantages because it is derived from a plant and further satisfies the necessity for practical use. Further, the aryl acylamidase gene is
Since it is originally a herbicide anti-resistance gene, it can be applied with higher selection pressure than conventional selection markers, and is expected to be used as a selection marker gene for more excellent genetic engineering.

[0007] On the other hand, besides use as a marker gene, an aryl acylamidase gene imparts DCPA resistance to a plant or the like by introducing it into the plant or the like,
It is expected that it can be used to create herbicide-resistant plants and the like.

To solve these problems, enzymatic purification of arylacylamidase was performed from tulip and cloning of the gene was attempted. It has been desired to provide an arylacylamidase gene having high activity. In addition, since tulips are not generally regarded as food crops, if possible, a gene derived from a plant that is widely used for food is desirable as a selectable marker gene for genetic engineering. Therefore, an object of the present invention is to produce a parsley aryl acylamidase gene based on the assumption that parsley aryl acyl amidase is a new type of enzyme having higher activity than tulip aryl acyl amidase. It is used as a selectable marker for genetic engineering, and in creating a gene for producing a herbicide-tolerant plant.

[0009]

Means for Solving the Problems As a result of repeated studies for solving the above-mentioned problems, the present inventors have succeeded in completely purifying parsley-derived arylacylamidase, and have found that the activity of this parsley-derived arylacylamidase is It has been found that it has an activity about 6.5 times higher than the activity of tulip-derived arylacylamidase which is considered to be the most active among known ones. The analysis of the partial amino acid sequence of the arylacylamidase protein derived from parsley and the production of an antibody were successful. Using these, the cloning of parsley arylacylamidase was successful, and the analysis of the base sequence and the like was also successful.

That is, the first aspect of the present invention relates to a gene having a base sequence encoding the amino acid sequence of a parsley-derived arylacylamidase shown in FIG.

[0011] The second aspect of the present invention relates to a vector containing the DNA strand.

The nucleotide sequence encoding the amino acid sequence of the arylacylamidase of the present invention is shown in FIGS. Since this base sequence does not fit into one figure in the electronic application, the entire sequence is described separately in FIGS.

The gene of the present invention, that is, the DNA chain, can be obtained as follows. First, purify the aryl acyl amidase from parsley, especially parsley leaves and buds,
Rabbits are injected to obtain antiserum. Next, the amino acid sequence is partially determined by analyzing the arylacylamidase protein. Next, using the obtained antiserum as a probe, the gene (DNA chain) of parsley-derived arylacylamidase is cloned.

The second vector containing the parsley-derived arylacylamidase gene of the present invention can be prepared by preparing a c-DNA library from parsley m-RNA or genomic live A rally can be prepared and isolated by screening by an immunodetection method using a parsley-derived arylacylamidase antiserum.
Alternatively, it can be isolated from the above-mentioned c-DNA library or genomic library by screening using a oligonucleotide synthesized from the nucleotide sequence shown in FIGS. it can. In addition, the parsley-derived arylacylamidase gene can be prepared from parsley nuclear DNA or parsley c-DNA by using a synthesized oligonucleotide as a primer in accordance with a known conventional method based on the data shown in FIGS. Melization chain reaction method (abbreviated as PCR method: Science)
e. 230, 1350-1354, 1985).

An organism into which the gene has been incorporated can be produced as follows. That is, the parsley-derived arylacylamidase gene of the present invention is transformed into a suitable plant expression vector, for example, PBI121 (manufactured by Clontech), BIN19 (Nucleic, Acid, R).
essearch, 12, 8711-8721, 1982
), It is possible to construct an expression vector for producing a parsley-derived arylacylamidase in a plant or the like. The expression vector constructed by linking the arylacylamidase gene derived from parsley in this manner is, for example, transformed into a plant by Agrobacterium, for example, tobacco, potato, arabidopsis, asparagus, tomato, etc. When introduced, DCPA resistance can be imparted to plants and the like, and a herbicide-resistant crop can be produced. Further, those in which a suitable promoter, for example, a cauliflower mosaic virus 35S promoter, a nopaline synthase promoter, or the like, is linked to an arylacylamidase derived from parsley, for example, by an electroporation method, for example, a particle gun method, for example, soybean , Rice, corn, and the like to give DCPA resistance to plants and the like. In addition,
A gene to be introduced into a plant or the like is linked to a parsley-derived arylacylamidase, and the gene is introduced into a plant or the like using the same method as described above. The gene can be used as a drug selection marker gene to select only transformed plants and the like.

[0016]

【Example】

Production Example 1 Cloning of an arylacylamidase gene derived from parsley Step 1 Purification of an arylacylamidase derived from parsley A 30 mM phosphate buffer pH 7.0 was added to the leaves of parsley,
Grinding was performed with a polytron type homogenizer. Cell debris was removed by centrifugation at 8,000 rpm for 10 minutes in a high-speed cooling centrifuge. Ammonium sulfate was added to the supernatant to a 40% saturation concentration, and the precipitate was removed by centrifugation at 8,000 rpm for 10 minutes. Ammonium sulfate was added to the supernatant to a 60% saturation concentration, and the mixture was centrifuged at 8,000 rpm for 10 minutes to collect the precipitate, and 30 mM phosphate buffer (pH 7.0) was added to dissolve the supernatant.

The solution of the crude protein thus obtained is
Purified in step. 1. Removal of low molecular substances by dialysis. 2. 2. diethylaminoethyl cellulose chromatography Hydrophobic chromatography with Toyopearl HW55F (trade name) This step is a very specific process and utilizes the phenomenon that elution of most of the arylacylamidase is delayed under a high salt concentration. This step is almost pure. 4. 4. Hydroxyapatite chromatography Hydrophobic chromatography with butyl toyopearl (trade name) These steps 4 and 5 are for further removing remaining trace impurities.

The purified protein was subjected to SDS polyacrylamide electrophoresis and TSK gel G by the method of Laemmli (Nature, 227.680, 1970).
It was confirmed to be pure by high performance liquid chromatography using 3000SW (manufactured by Tosoh Corporation). When the enzyme activity was measured using this sample, 1.2 mg / mg was measured.
It showed a specific activity of 5 units. This activity is about 6.5 units compared to 0.19 units per mg of tulip.
It was twice as strong. Enzyme activity was measured by methods such as Kodama Osamu and Akatsuka Tadami (Faculty of Agriculture, Ibaraki University, 49
Vol. 22, p. 1973).

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 parsley-derived arylacylamidase protein to perform limited degradation. Thereafter, protein fragments were separated by SDS polyacrylamide electrophoresis, and this was electrophoretically transferred to Immobilon (manufactured by Millipore) on a synthetic resin membrane. In addition, TPCK trypsin (manufactured by Cooper Chemicals), which is a proteolytic enzyme, was added to the purified parsley-derived arylacylamidase protein to perform limited degradation. The peptide fragment was separated and purified by high performance liquid chromatography. Using these as samples, about 40 samples were tested for amino acid sequence by Edman degradation using an ABI model 477A pulse liquid protein sequencer. Amino acid sequence information was obtained from about 10 samples.

Step 3 Preparation of Antiserum Rabbit anti-arylacylamidase serum against purified arylacylamidase derived from parsley was prepared by the following method. 1.0 ml of a 1.0 mg / ml concentration of a parsley-derived arylacylamidase solution (30 mM, pH
7, Phosphate buffer solution / solution)
Unt) Rabbits were injected as antigens as suspensions and emulsified with complete adjuvant (manufactured by Difco), and the same procedure was repeated 4 weeks later, and 40 weeks later,
ml of blood was collected. Then, the obtained blood was left in a refrigerator for 2 hours, and this was centrifuged at 7,000 rpm for 1 hour.
After centrifugation for 5 minutes, anti-aryl acylamidase serum was obtained as a supernatant. This antiserum had sufficient titer to detect aryl acylamidase spotted at a concentration of 100 pg / mm ² on a nitrocellulose membrane at a 1000-fold dilution, but reacted strongly with E. coli proteins. Antiserum affinity purification was performed using a solid phase in which the acylamidase protein was immobilized on a nitrocellulose membrane. As a result, components reactive with the E. coli protein were removed, and an antibody suitable for gene cloning was obtained. This anti-arylacylamidase antibody solution was stored at 4 ° C. and used for gene cloning.

Step 4 Cloning of Aryl Acyl Amidase from Parsley (a) Preparation of Total RNA The instruments used in the following steps for handling RNA were all heat-treated at 250 ° C. for 4 hours or 2% diethyl pyrocarbonate. A solution treated with an aqueous solution and inactivated with an RNase was used. SDS from parsley buds
-The total RNA was adjusted using the phenol method. 40 g of a 90% phenol solution in 10 g of parsley buds, 1%
40 ml of the SDS solution was added, and the mixture was ground with a blender.
After centrifugation at 7,000 rpm for 10 minutes, the supernatant was taken out, and 9
20 ml of 0% phenol and 20 ml of chloroform were added, mixed, and centrifuged at 7,000 rpm for 10 minutes. 0.5 ml of 10 M lithium chloride and 10 ml of ethanol in the supernatant
And left at -20 ° C for 1 hour, 7,000 rotations, 10
After centrifugation for 5 minutes, the precipitate was dissolved by adding 5 ml of water.
5 ml of 10M lithium chloride was added, left at -20 ° C for 1 hour, and centrifuged at 7,000 rpm for 10 minutes. This operation was further repeated once, and then washed three times with 70% ethanol to obtain a total RNA fraction. This entire operation was performed 9 times, and each obtained total RNA was subjected to electrophoresis by agarose gel electrophoresis to select a high-quality product having a low content of degradation products, and to obtain a poly-containing m-RNA of the next step. (A) Used as a material for adjusting RNA.

(B) poly (A) containing m-RNA
Preparation of RNA From this total RNA, the method of Aviv and Leder (Pr
received of National Acad
emy of Science U.S.A. S. A. 69,1
408, 1972), using an improved m-RNA purification kit manufactured by Pharmacia,
The operation was performed according to the manual attached to this kit.
That is, purification of the poly (A) RNA fraction by oligo dT cellulose column chromatography. An outline of the operation will be described below. Total RNA is 0.5M KC
10 mM Tris-HCl buffer (pH
Using 7.5) as a binding buffer, m-RNA is bound by passing through a simple centrifugation column of oligo dT cellulose,
Then, after washing the column, m-
By eluting RNA, poly (A) RNA
I got

(C) Preparation of c-DNA Library cDNA synthesis from poly (A) RNA
Ler and Hoffmann's method (Gene, 25, 2).
63-269, 1983), which is an improved version of "cDNA synth" manufactured by Pharmacia.
The operation was carried out using an “esis kit” according to the manual attached to the kit. The outline of the operation is described below. 5 μg of poly (A) RNA obtained from parsley
To 20 μl with water. This sample is
After heating for 10 minutes, cool to 0 ° C. A first-strand synthesis reagent (mainly composed of reverse transcriptase, oligo dT primer, and dNTPs) is added and reacted at 37 ° C. for 1 hour.
Second-strand synthesis reagent (RNaseH, DNA polymerase)
Rasel and dNTPs are the main components. ) And 12
The reaction is carried out at 1 ° C. for 1 hour and then at 22 ° C. for 1 hour. Add 1 unit of Klenow fragment of DNA polymerase, add 3 units
Incubate at 7 ° C for 30 minutes. Add 50 μl of chloroform and 50 μl of phenol and stir. The reaction solution is centrifuged at 15,000 rpm for 1 minute in a microcentrifuge, and the supernatant is applied to a gel filtration column of Sephacryl S200. 5 μl of EcoRl adapter and 1 μl of ATP solution were added to the eluate.
4 Add 3 μl of DNA ligase and react at 12 ° C. overnight. Heat at 65 ° C for 10 minutes and cool to 0 ° C. 10
Add 1 μl of ATP solution and 1 μl of T4 polynucleotide kinase and react at 37 ° C. for 30 minutes. Add 50 μl of phenol and 50 μl of chloroform and stir. The reaction solution is centrifuged at 15,000 rpm for 1 minute in a microcentrifuge, and the supernatant is applied to a simple centrifugal gel filtration column of Sephacryl S200. The eluate is the synthesized cDNA. The volume of this eluate was about 150 μl. Of these, 10 μl was analyzed by 1% agarose electrophoresis after ribonuclease treatment, and it was confirmed that c-DNA was synthesized.

Among the cDNAs thus obtained,
20 μl was ligated to λ-gt11 phage DNA (Stratagene) treated with EcoRl and alkaline phosphatase. c-DNA and λ-gt11 DN
The ligation of A was performed with D4 by T4 phage DNA ligase.
Ligation was performed using a "DNA ligation kit" (manufactured by Takara Shuzo) based on the ligation reaction of NA. The reaction procedure is described in the “Nucleic Acids res
search, 14, 7617-7631, 1986
Years' method.

[0025] One-fifth of the thus obtained c-DNA-linked λ-gt11 was used for reassembly into phage particles (packaging). In this operation, packaging into phage particles was performed using "Gaigapack Gold", a kit manufactured by Stratagene. Thus, the λ-incorporated c-DNA
The gt11 phage was reconstructed and used as a cDNA library. This packaging operation was performed four times. As a result of counting by the dilution method, this DNA library contained about 10 million independent clones in total. In addition, it was soldered that the ratio of the recombinants was 90% or more and could be sufficiently used for cloning.

(D) Cloning The cloning of the arylacylamidase gene from the λ-gt11 c-DNA library was carried out by a conventional immunoscreening method (Young and Davis, Proceeding of Natio).
nal Academy of Science US
A 80, 1194-1198, 1983). That is, this library is used together with the host E. coli Y1090,
After plating on phage plaques to form 5,000 clones per plate, nitrocellulose membrane (Nitroplus 2000; Micron
Separation). This nitrocellulose membrane is coated with a TBSM solution (2% skim milk and 100%
30 mM Tris-HCl buffer pH containing mM NaCl
8.0), and then treated with a rabbit arylacylamidase antiserum diluted 1,000 times with a TBSM solution for 30 minutes. Next, the nitrocellulose membrane is washed and TBSM
Peroxidase binding 2 diluted 3,000-fold with solution
Next antibody (Bio-Rad Laboratory) 30
Minutes. Using Konica Immunostain (manufactured by Konica) as a coloring reagent, the expression vector λ-gt1 was used.
The protein produced by one phage was screened using antiserum to obtain a λ-gt11 phage having the target gene. Of about 10 million phages obtained in (b), about 3 million phages
Screening was performed using a rabbit arylacylamidase antiserum as a probe, and about 25 positive clones were obtained. 10 of these positive clones
The cut pieces were cut with a restriction enzyme EcoRI (manufactured by TOYOBO), and the inserted c-DNA was analyzed by agarose gel electrophoresis. As a result, 5 pieces were inserted with approximately the same length of about 1.3 kb to 1.4 kb. Since it contained c-DNA,
Further, a full-length clone was searched for these nine by direct partial base sequence determination and PCR, and the entire base sequence of the longest clone was determined.

(E) Determination of base sequence of cloned arylacylamidase and corresponding amino acid sequence The base sequence of cloned arylacylamidase was determined using AB using M13 phage and a fluorescent substrate.
This was carried out using a dye terminator kit of Company I and a DNA sequencer of ABI. For determination of the nucleotide sequence, the arylacylamidase c-DNA obtained in Production Example 1, step 4 (d) was inserted and ligated into phage PUC19 (Nippon Gene) and propagated in E. coli JM103 (Nippon Gene). . To create a deletion mutant for determining the entire nucleotide sequence, the kilosequence method was used. Using the deletion kit for kilosequence (manufactured by Takara Shuzo), the method of Henikoff (GENE, 2
8, 351-359, 1984), a group of DNAs having a portion of the arylacylamidase c-DNA fragment deleted from the end was prepared, and these were used as ABI DNA
The nucleotide sequence was determined using a sequencer. By linking these sequences, the entire nucleotide sequence was determined. DNA
Nucleotide sequence analysis computer analysis software DNAS
Analysis was performed by IS (Hitachi Software Engineering). Thereby, the obtained gene (DNA
As a result of the structural analysis of the chain, a base sequence encoding the amino acid sequence obtained from the protein analysis is included, which means that the obtained gene (DNA chain) is a parsley-derived arylacylamidase gene. Was confirmed. Moreover, when compared with the c-DNA base sequence of tulip arylacylamidase, it is a gene having no significant similarity in base sequence, and it is consensus that tulip arylacylamidase and parsley arylacylamidase have DCPA degrading activity. However, it was proved that the gene was not closely related. A part (about 80%) of this c-DNA is translated into amino acids to form an enzyme. This amino acid translation region is called an open reading frame, and has the amino acid sequence shown in FIG.

[0028]

Industrial Applicability The gene of the present invention has a degrading activity of herbicide DCPA in a host, especially in a plant or the like. Therefore, in gene recombination technology, it can be used as a drug selectable marker gene derived from food crops, or as a herbicide resistant gene. It can be used effectively for producing plants and the like. INDUSTRIAL APPLICABILITY The present invention mainly contributes to genetic engineering of plants as a safe herbicide resistance gene or a drug selection marker gene derived from food crops.

[Brief description of the drawings]

FIG. 1 shows a part of the base sequence of the c-DNA of parsley-derived arylacylamidase of the present invention.

FIG. 2 shows a part of the base sequence of the c-DNA of the arylacylamidase derived from parsley of the present invention, which is continued from FIG.

3 shows a part of the base sequence of the c-DNA of the arylacylamidase derived from parsley of the present invention, which is continued from FIG. 2.

FIG. 4 shows a translation of an open reading frame of c-DNA of an arylacylamidase derived from parsley of the present invention into an amino acid sequence.

Claims (3)

(57) [Claims] 1. A gene having a base sequence encoding the amino acid sequence of a parsley-derived arylacylamidase shown in FIG. 2. The gene according to claim 1, wherein the nucleotide sequence is shown in FIGS. 3. A vector comprising the gene according to claim 1 or 2.