JPH03290190A - Novel dna strand - Google Patents

Abstract

NEW MATERIAL:The DNA strand having a base sequence coding an amino acid sequence of tulip allylacylamidase. EXAMPLE:The cDNA having the base sequence of formula. USE:Preparation of a plant resistant to herbicide, etc. PREPARATION:Purified allylacylamidase separated from tulip is injected into a rabbit and the gene (DNA strand) of allylacylamidase of tulip is cloned by using the obtained antiserum as a probe.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention provides a cloned DNA of allyl acylamidase.
, fragments thereof, and plasmids into which the above-mentioned cloned DNA or fragments have been integrated, or plants, animals, and microorganisms.

[Conventional technology]

Propanil (3,4-dichloropropionanilide) is a herbicide used on rice. Weeds die, but rice shows resistance. This is because rice possesses the enzyme allyl acylamidase, which specifically decomposes and detoxifies propanyl (Science, 160.13
60.1968).

Allyl acylamidase that decomposes propanyl is found in rice, parsley, and tulip (Phytochemi).
It is known that it is also contained in very limited plants such as A. stry, 11.521-527.

However, aryl acylamidase from higher plants has not been completely purified, and there is not much knowledge regarding this enzyme. Furthermore, no cloning of the allyl acylamidase gene has been performed.

On the other hand, in genetic engineering of plants, it is important to introduce a drug resistance selection gene into the plant by linking it to the desired gene in order to select only cells or plants into which the desired gene has been incorporated. Common. The kanamycin resistance gene is the most commonly used drug resistance gene for this purpose. However, since kanamycin and kanamycin resistance genes were not originally developed for genetic manipulation of plants, their performance is not very high, and selection may not be successful depending on the plant species. To compensate for these drawbacks, hygromycin resistance genes are also used, but in any case, they are antibiotic resistance genes derived from microorganisms, and these are not suitable for practical use in plants due to environmental and safety issues. Especially genes of food crops] are considered unsuitable for second science (Plant Cell Engineering, 1, 62. 1989)
,.

Furthermore, the scope of agricultural use of the herbicide propanil itself is limited, as most crops do not have aryl acylamidase activity, and it is mainly used for rice.

[Problem to be solved by the invention]

As selectable marker genes necessary for plant genetic engineering, genes cloned from plants and the like are considered more desirable than microbial genes, as they have fewer concerns about safety. Currently, the neomycin phosphoodorans-ferase n31 gene (NPTI
I) and the hygromycin phosphotransferase gene have the drawbacks mentioned above in the "Prior Art" section. Furthermore, the fact that the drugs to which resistance is caused by these genes are antibiotics is considered undesirable in itself, especially considering the application to food crops and the problem of environmental release. If the aryl acylamidase gene of higher plants can be cloned, it is expected that it will become a selectable marker gene that does not have these drawbacks and also meets the needs for practical use. Furthermore, since the aryl acylamidase gene is originally a herbicide-resistant plant, higher selection pressure than conventional selection markers can be applied to it, and it is also expected to have the advantage of being an excellent selection marker gene for genetic engineering.

On the other hand, in addition to its use as a marker gene, the allyl acylamidase gene can be used to impart propanyl resistance to plants by introducing it into plants, etc., and to create herbicide-resistant plants. It is expected that

[Means to solve the problem]

As a result of extensive research to solve the above-mentioned problem, the present inventor discovered that tulip allyl acylamidase is optimal, and has thus arrived at the present invention.

That is, the first aspect of the present invention relates to a DNA strand characterized by having a base sequence encoding the amino sequence of tulip allyl acylamidase. this DNA
The chain is the tulip allyl acylamidase gene.

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

The third aspect of the present invention relates to plants, animals, or microorganisms containing the above DNA strand.

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

First, allyl acylamida from tulips. -se was purified and injected into rabbits to obtain antiserum.

Next, the amino acid sequence of allyl acylamidase was partially determined by protein analysis. Next, using the obtained antiserum as a probe, the allyl acylamidase gene (DNA chain) was cloned from the tulip. As a result of structural analysis of the obtained gene (DNA chain), it was found that the obtained gene (DNA chain) contained a sequence encoding the amino acid sequence obtained from the protein analysis.
This confirmed that the A chain) was an allyl acylamidase gene.

The nucleotide sequence shown in FIG. 1 and the amino acid sequence shown in FIG. 3 are shown.

The second phage or plasmid containing the tulip allyl acylamidase gene of the present invention is obtained from c-D from tulip m-RNA according to known conventional methods.
Create an NA library or from nuclear DNA,
It can be isolated by creating a genomic library and screening by immunodetection method using tulip allyl acylamidase antiserum. Alternatively, from the above c-DNA library or genomic library,
The oligonucleotide synthesized from the base sequence shown in FIG. 1 can be used as a probe and isolated by screening according to a known conventional method. In addition, the tulip allyl acylamidase gene was prepared using oligonucleotides synthesized from the nucleotide sequence shown in FIG.
From NA, or Tulip c-DNA, the polymerization chain reaction method (abbreviated as PCR method: 5science, 230.1350
-1354.1985).

The plants and the like containing the DNA strand, which is the third aspect of the present invention, can be produced as follows.

The tulip allyl acylamidase gene of the present invention can be expressed in a suitable plant expression vector, such as PBI], 21 (
Nucleic), BIN19 (Nucleic), BIN19 (Nucleic
, Ac1d, Re5earch, 12.8711
-8721° (1982), etc., an expression vector for producing tulip allyl acylamidase in plants etc. can be constructed.

The expression vector constructed by ligating the allyl acylamidase gene in this way is introduced into tobacco, potato, Arabitopsis, asparagus, tomato, etc., for example, by Agrobacterium-based plant transformation method. Propanil resistance can be imparted to plants, etc., and herbicide-resistant crops can be created. Further, a suitable promoter, such as cauliflower mosaic virus 35S promoter, nopaline synthetase promoter, etc., linked to tulip allyl acylamidase can be used, for example, by electroporation method 1.
For example, by the particle gun method, it can be introduced into plants such as dice, rice, and corn, animals, or microorganisms to impart propanyl resistance to the plants. Furthermore, the gene to be introduced into plants, etc. is linked to tulip allyl acylamidase, and using the same method of gene introduction into plants, etc. as described above,
Once introduced, the aryl acylamidase gene expressed in plants etc. can be used as a drug selection marker gene to select only transformed plants etc.

〔Effect of the invention〕

Since the DNA strand of the present invention has the activity of degrading the herbicide propanil in a host, especially in plants, it can be effectively used as a drug selection marker in genetic recombination technology or in the production of herbicide-resistant plants. be able to.

[Examples, etc.]

Production Example 1 Cloning of allyl acylamidase gene Step 1 Purification of tulip allyl acylamidase 30mM phosphate buffer PI (7,0) was added to commercially available tulip bulbs (variety name Nirose Beauty), and the mixture was ground using a Polytron homogenizer. .

10. in a high-speed refrigerated centrifuge. The cell layer was removed by centrifugation on OQO rotation for 10 minutes. Ammonium sulfate was added to the supernatant to give a 40% saturation concentration, and the mixture was centrifuged at 10,000 rpm for 10 minutes to remove the precipitate. Ammonium sulfate was added to the supernatant to give a 60% saturation concentration, and the mixture was centrifuged at 10,000 rpm for 10 minutes to collect the precipitate.

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

1. Removal of low molecular weight substances by dialysis.

2. Diethylaminoethyl cellulose chromatography 3. Gel filtration chromatography using Toyopearl HW55F 4. Hydroxylapatite chromatography Purified proteins were purified using the Laemmli method (Natural
The purity was confirmed by SDS-polyacrylamide electrophoresis and high-performance liquid chromatography (Re, 227.680.1970).

Step 2 Determination of amino acid partial sequence.

Lysyl endopeptidase (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.), which is a proteolytic enzyme, was added to the purified allyl acylamidase protein for limited degradation. After that, protein fragments were separated by SDS polyacrylamide electrophoresis,
This was electrophoretically transferred to a synthetic resin membrane of Imopilone (manufactured by Millipore). Using this as a sample, using an ABI model 477A gas phase protein sequencer, Edma
The amino acid sequence was determined by n-resolution.

Step 3: Preparation of antiserum Rabbit anti-allyl acylamidase serum against purified allyl acylamidase was prepared by the following method. ]
,, Allyl acylamidase solution with Omg/mQ concentration (
30mM, pH 7, phosphate buffer/solution) 1.0m
A suspension of Q with an equal amount of Freund's complete adjuvant (manufactured by Difco) was injected into rabbits as an antigen, and 4 weeks later, the same procedure was carried out again.
A week later, 20 mfl of blood was collected. The obtained blood was left in the refrigerator for 2 hours, and then centrifuged for 7.
The mixture was centrifuged at 000 rpm for 15 minutes to obtain anti-allyl acylamidase serum as a supernatant. This antiserum had sufficient titer to detect allyl acylamidase spotted on a nitrocellulose membrane at a concentration of 1100 p/mm.

Step 4 Cloning of tulip allyl acylamidase (a) Preparation of poly (A) RNA containing nrRNA Total RNA was prepared from tulip bulbs using the SDS/phenol method. tulip bulb 1
40 mQ of 90% phenol solution and 40 mQ of 1% SDS solution were added to 0 g, and the mixture was ground with a blender. 10,0
After centrifugation at 10,000 rpm for 10 minutes, the supernatant was taken, and 20 mQ of 90% phenol and 20 mfl of chloroform were added and mixed, followed by centrifugation at 10,000 rpm for 10 minutes. Supernatant: 10M lithium chloride 0.5ml, ethanol 10mQ
was added, left at -20℃ for 1 hour, 10,000 rpm, 1
After centrifugation for 1-10 minutes, 5 mR of water was added to the precipitate to dissolve it. Add 5 mQ of 10M lithium chloride and heat to -20°C.

The mixture was left to stand for 1 hour and centrifuged at 10,000 rpm for 10 minutes.

Furthermore, this operation was repeated once more, and then washed three times with 70% ethanol to obtain a total RNA fraction.

From this total RNA, a poly (A) RNA fraction was purified by oligo dT cellulose column chromatography according to the method of Aviv and Leder (Pr
oceeding of National Acad
emy of 5scienceUSA 69,1408
．． (1972). That is, binding to oligo dT cellulose using 10mM 4ris-HCJ2 buffer (PH7,5) containing 0.5M KCQ as a binding buffer,
Next, pc+ly (A) RNA was obtained by elution with a buffer containing no KCQ.

(b) Creation of c-DNA library poly(A
) Synthesis of cDNA from RNA is performed by Gubler and ho
ffmann's method (Gene, 25.263-26
9. 1983), with improvements made thereto, by Pharmacia.
This was done using sis kit''2.

The operation was performed according to the manual included with this kit.

Provides an overview of operations. poly obtained from tulips
(A) Add water to 5 μg of RNA to make 20 μg. This sample was heated at 65°C for 10 minutes, and then O
Cool to 'C. A second strand synthesis reagent (mainly consisting of reverse transcriptase, oligo dT primer, and dNTPs) is added and reacted at 37°C for 1 hour. Second strand synthesis reagent (RNa
seH, DN Apolymerasel, dNTP
The main component is s. ) and react at 12°C for 1 hour, then at 22°C for 1 hour.

Add 1 unit of Flenow fragment of DNA polymerase and react at 37°C for 30 minutes. Add 50μ flood of chloroform and 50μ flood of phenol and stir. The reaction solution was centrifuged at 15,000 rpm for 1 minute in a microcentrifuge, and the supernatant was applied to a Sephacryl 5200 gel filtration column. Add 5μ of EcoR1 adapter and 1μQ of ATP solution to the eluate.
Add 3μ of T4 DNA ligase and react overnight at 12°C. Heat at 65°C for 10 minutes and cool to 0°C. 1
Add 0μα ATP solution and 1μα T4 polynucleotide kinase, and react at 37°C for 30 minutes. 5oμ
Add α phenol and 50μ flooded chloroform and stir. The reaction solution was centrifuged at 15,000 rpm for 19 minutes using a microcentrifuge, and the supernatant was applied to a Sephacryl S 200 gel filtration column. The eluate is the synthesized cDNA.

By performing the above operations, cDNA was synthesized using 5 μg of tulip poly(A) RNA.

The cDNA thus obtained was ligated to λ-gtll phage DNA (manufactured by Stratagene) that had been treated with EcoRl and alkaline phosphatase. c.
-DNA and λ-gt11'-DNA are connected by T4
- Based on the DNA ligation reaction using Phage DNA ligase, ligation was performed using a DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.). The reaction operation is performed using the “Nuc” instructions provided in this kit.
leicacldsresearch, 14,
7617-7631, 1986, according to the method of H.

λ-gt ligated with the c-DNA thus obtained
Of the 175 liters, 175 were used for packaging into phage particles. In this operation, packaging into phage particles was performed using a kit "Gigapack Plus" manufactured by Stratagene. In this way, c − D
The λ-gtll phage in which NA had been integrated was reconstituted, and this was used as a cDNA library. this DNA
The library contained 1 million clones.

(C) Cloning The aryl acylamidase gene was cloned from the c-DNA library of λ~gtll by a conventional immunoscreening method (Young and Davjs, Proceeding of Nation
al Academy of Scj, ence US
A 80, 11941198.1983). That is, this library was used together with host E. coli Y1090 for 1
Plates were plated on 10 plates at a rate of 5,000 clones per plate, and after phage plaque formation was transferred to a 21-cellulose membrane using Troplus 2000; Micron 5- Separation Company H). . This nitrocellulose membrane was soaked in a TBSM solution (2% skim milk and 100%
30mM Tris-HCl buffer pH containing mM NaCl
After treatment with 8,0), 1% with TBSM solution and 30% with rabbit allyl acylamidase antiserum diluted 100 times.
Processed for minutes. Next, wash the nitrocellulose membrane with TB
It was treated for 30 minutes with peroxidase-conjugated antibody 2 (manufactured by Bio-Rat Laboratories) diluted 3,000 times with SM solution. Using Konica Immunostin (manufactured by Konica) as a coloring reagent, the expression vector λ-
The protein produced by gshi11 phage was screened using antiserum and λgtl containing the target gene was found.
l phage was obtained. Of the approximately 1 million phages obtained in (b), approximately 50,000 phages were screened using rabbit aryl acylamidase antiserum as a probe, and approximately 18 positive clones were obtained. Twelve of these positive clones were cut with the restriction enzyme EcoRI (TQYOB (manufactured by 1 company) and analyzed on an agarose gel). As a result of analyzing the inserted c-DNA by electrophoresis, 9 clones were found to be approximately 1.3 kb to 1. Since the inserted c-DNA contained approximately the same length of .4 kb, these 9 DNAs were further cut and analyzed using the restriction enzyme HaeIII (manufactured by TOYOBO). Since the cleavage pattern was shown, the nucleotide sequence of one of the clones was determined.

(d) Base sequence determination of cloned allyl acylamidase and corresponding amino acid sequence The base sequence determination method of cloned allyl acylamidase was performed using an ABI kit using M13 phage and fluorescent primers and an ABI DNA sequencer. went. Manufacturing example step 4 for determination of base sequence
Allyl acylamidase c-D obtained by (c)
The NA was inserted and ligated to phage M], 3 II + p 18 (manufactured by Tupon Gene) and propagated in Escherichia coli J old 03 (manufactured by Tupon Gene). A kilosequencing method was used to create deletion mutants for determining the entire base sequence. Using a deletion kit for kilosequencing (manufactured by Takara Shuzo Co., Ltd.), Hen1koff's method (GENE, 2) was used.
8, 351-359 (1984), a portion of the DNA was prepared by deleting a portion of the aryl acylamidase c-DNA fragment from the end, and the base sequence was determined using an ABI DNA sequencer. . The entire base sequence was determined by concatenating these sequences.

Figure ↓ shows tulip allylacylamidase c.
- Shows the entire base sequence of DNA. FIG. 2 shows the sequencing strategy used to determine the base sequence of the tulip aryl acylamidase gene.

FIG. 3 shows the amino acid sequence corresponding to the DNA base sequence of allyl acylamidase determined in this manner.

Production Example 2 Production step I of aryl acylamidase gene integration plasmid Preparation of Ti vector fragment Plasmid P
B l121 (manufactured by TOYOBO) with restriction enzyme Xbal
and EcoRI (manufactured by TOYOBO).
The lkb fragment was electrophoretically separated and collected using low melting point agarose (manufactured by Nacalai Tesque).

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

1 obtained by the operation of production example step 4 (C),
4kb of allyl acylamidase c-DNA was inserted into plasmid PUc18 (Tupone gene) and dam
It was grown in Escherichia coli GM33, which is a minus bacterium. The plasmid obtained by the above procedure was transformed into PUc18/aa
It was named a.

(b) PUc18/aaa with restriction enzyme EcoRI and Bc
1,1 (manufactured by TOYOBO), and the 0.3 kb fragment was electrophoretically separated and collected using low melting point agarose (manufactured by Nacalai Teks). Furthermore, a 0.3 kb fragment was extracted with the restriction enzyme Mn.
j 1, and a 0.17 kb fragment was electrophoretically separated and collected using low melting point agarose (manufactured by Nacalai Tesque).

9- (b) PUc18/aaa with restriction enzyme Bcl 1 and Sm
The fragment was cut with aI (manufactured by TOYOBO), and the 3.8 kb fragment was electrophoretically separated and collected using low melting point agarose (manufactured by Nacalai Teks).

(c) Join the two fragments of (a) and (b) above,
Escherichia coli 1 (grown in BIOI. The plasmid obtained by the above procedure was transformed into PUc/aaa-de], 6
It was named 0. PUc/aaa-del60 was cut with restriction enzymes Xbal and EcoR], and the DNA fragment from which approximately 60 residues of the 5' end of the allyl acylamidase DNA had been removed was placed in low melting point agarose (manufactured by Nacalai Tesque).
It was electrophoretically separated and collected and named aaa-del60.

Step 3 Integration of the aryl acylamidase gene into the T'' vector.

PBII21 fragment and allyl acylamidase gene aaa-del6 obtained in step 1 and step 2
0 was ligated to obtain plasmid PKAAA.

This plasmid is designed to express aryl acylamidase activity in plants. Therefore, propanil resistance can be imparted to the introduced plants (see Figure 4).

[Brief explanation of the drawing]

Figure 1 shows the cD of tulip allyl acylamidase.
The entire base sequence of NA is shown. The part that codes for the enzyme protein of allyl acylamidase is the 6th part of the base sequence.
It has a coding region of 1047 bases starting from the fourth ATG (start codon) and ending with the stop codon TGA, and codes for 349 amino acids. Figure 2 shows tulip aryl acylamidase (1, DN
The restriction enzyme map of A is shown. The white arrow indicates the protein coding region of aryl acylamidase. The black arrows indicate the range that was sequenced at each time during sequence determination. The numbers indicate the number of base residues of the nucleic acid from the 5' end. FIG. 3 shows the amino acid sequence corresponding to the base sequence of tulip aryl acylamidase cDNA, and FIG. 4 shows the physical map of plasmid PKAAA. AAA...Allyl acylamidase gene CaMV
35SP...353 of cauliflower mosaic virus
Promoter・DNA NPTII・・Neomycin phosphorol-transferase 2・Gene RB・・Ti plasmid light border region・DNA
LB...Ti plasmid left border region/DNA
3-10 TTGTTCTGTG 0 GGGGAGGAAG 10 AGCCGCCGGC 60 TCTGTAGTTTC 10 CGACGAAATC 60 GACGCATCGA 10 GTGGCCACCA 60 CCTCTACCTC 10 0 GT CCGCCGCC 0 GGTATGGTCC 20 TCATGCCTGC 70 CCACCAATTC 20 GAGTTGGACC 70 GCGTCTCAA 20 AAGACGTCAT 70 CCCAACGTCA 20 3゜CGCTGTATCT 0 GCAGCAAGGA 30 CGTCTTATAG 80 ATCTCTCTCG 30 TAAGCCCTT 80 GGCACCACCG 30 CATTGACCCC 80 GTCGACTTAC 30 0 TTTGGATGCA 0 TATCGTCCGA 40 GGTGGAATAA 90 CCGTCGCCCA 40 CTTGATCATT 90 TCATCCCGGC 40 GCCACCGG CG 90 CGTCAAGAAG 40 0 AAGATGGGCG 00 CAGACGGTCA 50 ATATCCTGGT 00 CAGCTTTAGA 50 TACAAGGATG 00 CTGCCCCGAG 50 TCTCAGTGCG 00 CTC CCCGTTC 50 ω Fufu ＞ ＞Φ−−Φ U(1))-10J(Jg0)ωOL,>ko)
-0 工の” 0 ” 工”ItlLG)
(Do) ω 0 Ko of Meniko on Hus Island [>
L's Φ ψJ
(L J ＞ ＞ J ＜−−
Ω, Shi >-'s OCo LO Ko >Of150〕QC (Me L - Tameichito's > - ←) Φ0 Shiba "Kuku L 0 Tsurigrig"To>

Claims (1)

[Scope of Claims] 1. A DNA strand characterized by having a base sequence encoding the amino acid sequence of tulip allyl acylamidase. 2. A plasmid containing the DNA strand according to claim 1. 3. A plant, animal or microorganism comprising the DNA strand according to claim 1.