CN100395265C

CN100395265C - Soybean WRKY transcription factor GmWRKY6 and its coding gene and use

Info

Publication number: CN100395265C
Application number: CNB2005100052064A
Authority: CN
Inventors: 陈受宜; 张劲松; 田爱国; 何锶洁; 杜保兴
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2005-02-01
Filing date: 2005-02-01
Publication date: 2008-06-18
Anticipated expiration: 2025-02-01
Also published as: CN1814620A

Abstract

The present invention discloses a transcription factor GmWRKY6 of a soybean WRKY, a coded gene thereof and application thereof. The aim of the present invention is to provide a transcription factor GmWRKY6 of a soybean WRKY, a coded gene thereof and an application for the transcription factor GmWRKY6 in stress resistant plant variety breeding. The transcription factor of a soybean WRKY is a protein with one of the following amino acid residue sequences: 1) SEQ ID No. 1 in a sequence list, and 2) a protein for regulating and controlling the stress resistance of a plant, which is used for substituting, deleting or adding one to ten amino acid residues of an amino acid residue sequence of SEQ ID No. 1 in the sequence list and has a transcriptional activation function. GmWRKY1 of the present invention is used for breeding stress resistant plant varieties, especially stress resistant soybean varieties, and has important meanings for increasing agronomic crop yield, especially soybean yield.

Description

A kind of soybean WRKY transcription factor GmWRKY 6 and encoding gene thereof and application

Technical field

The present invention relates to the WRKY class transcription factor relevant and encoding gene and application in the plant genetic engineering field, particularly derive from the WRKY class transcription factor relevant and encoding gene and its application in cultivating the plant with adverse resistance kind of soybean with resistance of reverse with resistance of reverse.

Background technology

The variation of physics, chemical factor in the environment, the factors of coercing such as for example arid, saline and alkaline, low temperature have material impact to growth and development of plant, can cause the extensive underproduction of farm crop when serious, therefore cultivating the high crop of resistance of reverse is one of major objective of plant husbandry.At present, using gene engineering technique carries out breeding and has become one of important method that improves the crop resistance of reverse.Higher plant cell has many approach and replys various environment stresses in the environment, and wherein transcription factor plays a part the regulation and control effector of anti-the retrocorrelation and expresses.Now in plant, found the multiclass transcription factor relevant, for example: the DREB class among the EREBP/AP2, bZIP, MYB or the like with plant stress tolerance.WRKY is the plant specific transcription factor, and the characteristics of this class transcription factor are to have 1-2 WRKYGQK conserved domain and 1-2 class zinc finger element.Only report that so far it takes place the defence of pathogenic bacteria, aging, form with plant and relevant to replying of injury etc., does not know also whether it is relevant to the patience of abiotic stress with plant.

Soybean is important oil crops, also is the main source of plant protein, understands fully its anti-contrary mechanism, and then improves its resistance of reverse, has important theory and realistic meaning.

Summary of the invention

The purpose of this invention is to provide a kind of WRKY class transcription factor and encoding gene thereof relevant that derives from soybean with resistance of reverse.

Soybean WRKY class transcription factor provided by the present invention, name is called GmWRKY6, derives from Glycine soybean (Glycine max (L.)), is the protein with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 1;

2) with SEQ ID № in the sequence table: 1 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the protein of the regulation and control stress resistance of plant of transcriptional activation function.

Wherein, sequence 1 in the sequence table is made up of 184 amino-acid residues, from the 112nd-118 amino acids residue sequence of aminoterminal (N end) is the WRKY conserved domain of WRKY class transcription factor, is zinc fingers from the 122nd-153 amino acids residue sequence of aminoterminal.

The encoding gene GmWRKY6 of above-mentioned soybean WRKY class transcription factor is one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 2 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 1 protein sequence;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 2 dna sequence dnas hybridization that limit.

The rigorous condition of described height can be at 0.1 * SSC, in the solution of 0.1%SDS, washes film under 65 ℃.

SEQ ID № in the sequence table: 2 by 917 based compositions, and its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence from 5 ' end the 34th to the 588th bit base; From 5 ' end the 367th to the 387th bit base is the encoding sequence of WRKY structural domain conservative among the GmWRKY6; From 5 ' end the 427th to the 522nd bit base is the encoding sequence of zinc fingers; From 5 ' end the 589th to the 917th bit base is non-translational region.The expression of GmWRKY6 is subjected to inducing of ethene, low temperature, high salt and arid, and with plant defence, aging, the form of pathogenic bacteria is taken place and relevant to replying of injury etc.

Contain expression carrier of the present invention, clone and host bacterium and all belong to protection scope of the present invention.

Arbitrary segmental primer is to also within protection scope of the present invention among the amplification GmWRKY6.

Utilize plant expression vector, the encoding gene importing vegetable cell with GmWRKY6 of the present invention can obtain environment stress tolerance enhanced transgenic cell line and transfer-gen plant.

When using GmWRKY6 to make up plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or inducible promoter.For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, as adding selected marker's (gus gene, luciferase genes etc.) that can in plant, express or antibiotic marker thing (gentamicin marker, kantlex marker etc.) with resistance.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

Carry GmWRKY6 of the present invention plant expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and the plant transformed tissue cultivating is become plant.By the plant transformed host both can be monocotyledonss such as paddy rice, wheat, corn, also can be dicotyledonss such as cucumber, tomato, willow, turfgrass, lucerne place.

GmWRKY6 of the present invention is to cultivating the particularly anti-contrary soybean varieties of plant with adverse resistance kind, and particularly the output of soybean is significant to improve farm crop.And the regulation and control that the certain involved in plant of some members from molecular biology angle proof WRKY family is replied abiotic stress, it is expressed and the anti-abiotic stress of plant is proportionate.

The present invention will be further described below in conjunction with drawings and Examples.

Description of drawings

Figure 1A-Fig. 1 E is the expression characterization of GmWRKY6 under abiotic stress is coerced

Fig. 2 is the structure of GmWRKY6 in the soybean gene group

Fig. 3 is the structure synoptic diagram that contains the plant expression vector of GmWRKY6

Fig. 4 A-Fig. 4 B for change GmWRKY6 Arabidopis thaliana and wild-type plant under salt stress with the normal growth condition under growth fraction

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment.

The screening of embodiment 1, soybean GmWRKY6 encoding gene and the clone of cDNA thereof

1) carry out BLAST retrieval in the soybean est database, cluster to 64 comprises the WRKY genoid fragment of complete WRKY structural domain, according to 64 pairs of primers of dna sequence dna design of these 64 WRKY;

2) with ordinary method from respectively to extract total RNA the seedling of 250mM NaCl, arid ,-4 ℃ of processing (treatment time was 3 weeks) and untreated (contrast) soybean varieties section rich 1;

3) the reverse transcription product with total RNA of the above-mentioned big bean seedlings of handling through different methods is a template, and respectively under the guiding of 64 pairs of primers, measure the expression of 64 WRKY and the relation of above-mentioned processing with the RT-RCR method, 20 μ l PCR reaction systems are: 1 μ l, one chain cDNA, each 1 μ l of 20 μ M upstream and downstream primers, 2 μ l 10X PCR damping fluids, 0.4 μ ldNTP (10mM) and 1U Taq archaeal dna polymerase, and with ultrapure water postreaction system to 20 μ l.The PCR reaction conditions is: 94 ℃ of 5min of elder generation; 94 ℃ of 1min then, 56 ℃ of 1min, 72 ℃ of 1min, totally 31 circulations; Last 72 ℃ are extended 10min.

Use primer 1:5 '-GTTTTCATTTACTACCATATTTA-3 ' and primer 2: 5 '-CTAATACAAGATACTTACTT-3 ' can be from the identical gene that through the soybean material of 250mM NaCl and drying treatment, increases, and under the same conditions from increasing less than this gene through-4 ℃ of processing and untreated soybean material, show that this expression of gene is induced by arid and high salt obviously, suppressed by low temperature, with its called after GmWRKY6.This gene is checked order, sequencing result shows that GmWRKY6 has the nucleotide sequence of sequence 2 in the sequence table, by 917 based compositions, its encoding sequence is that coding has the protein of the amino acid residue sequence of sequence 1 in the sequence table from 5 ' end the 34th to the 588th bit base.The WRKY conserved domain that is WRKY class transcription factor from the 112nd-118 amino acids residue of aminoterminal in the sequence 1 is a zinc fingers from the 122nd-153 amino acids residue of aminoterminal.GmWRKY6 is a WRKY class transcription factor.

Similar proteic comparison in embodiment 2, GmWRKY6 and the plant

The amino acid residue sequence of GmWRKY6 is carried out similarity relatively with similar albumin A TWRKY50 (AAL61857), ATWRKY51 (AAL29429) of Arabidopis thaliana and soybean and the amino acid residue sequence of ATWRKY59 (AAL50786), the result is as shown in table 1, shows that the similarity of GmWRKY6 and ATWRKY50, ATWRKY51 and ATWRKY59 is respectively 39.3%, 38.9% and 29.2%.

Table 1 GmWRKY6 and similar proteic similarity and diversity percentage ratio

	ATWRKY50	ATWRKY51	ATWRKY59	GmWRKY21	GmWRKY	6
	ATWRKY50	ATWRKY51	ATWRKY59	GmWRKY21	GmWRKY	6	ATWRKY50	37.6％	31.2％	39.3％	39.3％
ATWRKY51			28.9％	37.6％	38.9％		ATWRKY50	37.6％	31.2％	39.3％	39.3％
ATWRKY51			28.9％	37.6％	38.9％	ATWRKY59			28.9％	29.2％

Embodiment 3, the expression characteristic of GmWRKY6 gene under environment stress

The seed kind of soybean varieties section rich 1 in basin, after 2 weeks of growing, is carried out the following processing (treatment time was 3 weeks) of coercing respectively to seedling:

Arid is handled: soybean seedling is taken out the moisture that blots on the root from soil, place on the exsiccant filter paper, arid is cultivated sampling after 0.5 hour, 1 hour, 3 hours, 6 hours and 12 hours under illumination condition respectively.

Salt is handled: the root system of soybean seedling is placed 250mM NaCl solution, take a sample after 0.5 hour, 1 hour, 3 hours, 6 hours and 12 hours in illumination cultivation respectively.

Ethene is handled: the root system of soybean seedling is placed ethene, take a sample after 0.5 hour, 1 hour, 3 hours, 6 hours and 12 hours in illumination cultivation respectively.

Subzero treatment: wheat seedling is placed 4 ℃ of incubators, take a sample after 0.5 hour, 1 hour, 3 hours, 6 hours and 12 hours in illumination cultivation respectively.

Osmotic stress is handled: the root system of soybean seedling places 20%PEG solution, takes a sample after 0.5 hour, 1 hour, 3 hours, 6 hours and 12 hours in illumination cultivation respectively.

Get each 1g of plant leaf of collection through above-mentioned different methods and the processing of different treatment time, place liquid nitrogen to grind, be suspended from the 4mol/L sulphur hydracid guanidine, and with acid phenol, chloroform extracting, in supernatant, add dehydrated alcohol and precipitate, will precipitate total RNA that obtains soluble in water at last.RNA with the different soybean samples that extract with aforesaid method is a template respectively, with ³²The cDNA of the GmWRKY6 of P-CTP mark is a probe, according to a conventional method the sample of handling through arid, high salt, ethene and osmotic stress is carried out Northern and analyzes; With the method for RT-PCR, under the guiding of primer 1 and primer 2, to carry out the expression analysis of GmWRKY6 through the sample of subzero treatment.The result shows that the expression of GmWRKY6 is induced by high salt, ethene and osmotic stress obviously shown in Figure 1A-Fig. 1 E, suppressed by low temperature (4 ℃).

Embodiment 4, the structure of GmWRKY6 gene in the soybean gene group

Extract the genomic dna of soybean varieties section rich 1 with ordinary method, with genomic dna restriction endonuclease Taq I, Hind III, EcoR I, EcoRV is after DraI and BamH I carry out complete degestion, to use ³²The cDNA of the GmWRKY6 of P-CTP mark is a probe, makes Southern and analyzes.The result shows that GmWRKY6 may exist with single copy as shown in Figure 2 in the soybean gene group.

The Function Identification of embodiment 5, GmWRKY6

1) genomic dna with soybean varieties section rich 1 is a template, under the guiding of primer 1 and primer 2, and pcr amplification GmWRKY6; 2) as shown in Figure 3, with the GmWRKY6 forward insert pBin438 (Li Taiyuan, Yingchuan, field, Qin Xiaofeng, Chinese science (B collects), 1994,24 (3), 276-282) between the BamH I of plant expression vector and the Kpn I restriction enzyme site; 3) with step 2) the plant expression vector arabidopsis thaliana transformation that contains GmWRKY6 that makes up.A transformed plant surplus the result obtains 10 is analyzed through Northern, shows that the expression amount of GmWRKY6 in wherein at least 3 strains is very high.The salt tolerance test shows after handling for 2 weeks with 250mM NaCl, place again under the normal condition and grow, (Fig. 4 A is the plant of handling through 250mM NaCl to the result shown in Fig. 4 A-Fig. 4 B, Fig. 4 B is not for there being the plant of coercing under the treatment condition), under nothing is coerced treatment condition, but transfer-gen plant and wild-type plant be normal growth all, and under hypersaline environment, the growing way of transfer-gen plant obviously is better than the wild-type plant, but show GmWRKY6 response abiotic stress really, it is expressed and the anti-abiotic stress of plant is proportionate.

Sequence table

<210>1

<211>184

<212>PRT

＜213〉Glycine soybean (Glycine max (L.))

<400>1

Met Asp Phe Tyr Phe Gly Asn Ser Pro Pro Tyr Pro Asn Asn Tyr Ala

1 5 10 15

His Asn Ser Leu Asn Met Ala Leu Ser Ser Pro Glu Ile Ala Leu Ser

20 25 30

Asp Tyr Leu Met Leu Asp Asp Tyr Val Asp His Gln Asp Ser Arg Ser

35 40 45

Ser Gln Ser Thr Glu Ser Ser Glu Lys Ala Thr Phe Ser Asp Ala Thr

50 55 60

His Gly Phe Ser Thr Gly Ala Thr Ser Lys Asn Asn Asn Ile Asn Cys

65 70 75 80

Lys Asn Gly Ile Ash Glu Asn Lys Gly Gly Val Gly Pro Arg Ile Ala

85 90 95

Phe Arg Thr Lys Ser Glu Leu Glu Ile Met Asp Asp Gly Tyr Lys Trp

100 105 110

Arg Lys Tyr Gly Lys Lys Ser Val Lys Ser Ser Pro Asn Leu Arg Asn

115 120 125

Tyr Tyr Lys Cys Ser Ser Gly Gly Cys Ser Val Lys Lys Arg Val Glu

130 135 140

Arg Asp Arg Asp Asp Tyr Ser Tyr Val Ile Thr Thr Tyr Glu Gly Val

145 150 155 160

His Asn His Glu Ser Pro Phe Thr Thr Tyr Tyr Ser Pro Ile Ser Phe

165 170 175

Val His Ser Asp Thr Thr Phe Lys

180

<210>2

<211>917

<212>DNA

＜213〉Glycine soybean (Glycine max (L.))

<400>2

gttttcattt actaccatat ttagttatca gcaatggact tctactttgg aaactctcct 60

ccttatccca ataattatgc tcataattct cttaatatgg ctctatcttc ccctgagatt 120

gcactatccg attatctcat gctcgatgac tatgttgatc atcaagattc tcgatcatca 180

caaagcaccg agtcatctga aaaagcaacc ttcagtgatg ccactcacgg attcagtact 240

ggtgcaacct ctaagaataa taacataaac tgcaaaaatg ggattaatga aaacaaaggt 300

ggagtgggtc caaggatcgc gtttagaacc aaatcagagc ttgagatcat ggatgatgga 360

tacaagtgga ggaagtacgg caagaagtcc gtgaagagca gtcccaatct aaggaactac 420

tacaaatgtt caagtggagg atgcagtgtg aagaaaaggg tggaaaggga tagagatgac 480

tacagctacg tgataacaac atatgaaggt gtgcacaatc atgagagccc atttaccaca 540

tactacagcc ccatctcctt cgtacattct gatactactt tcaaatgaca ccaacttcaa 600

acccttttgg gcatttacac ctccctagct taattatgtg ccaagaaaaa gcttggaaga 660

ttcgatcgag caggggaagc tatgtatcat tgcattgtct aatcaatcat aatttatttt 720

catgctgtaa taggttgaag agagtttcta tacctttttt tggttccacg tttaatatat 780

ctctcaatta aaaatcagcc tttgctagtt catgtaacaa ggaacatgat tctccatgtg 840

taaagttgtt caaatcttaa aagtaagtat cttgtattag gattagagta tctctcttgt 900

actctgttta ataataa 917

Claims

1. soybean WRKY class transcription factor, its amino acid residue sequence is shown in SEQ ID NO:1.

2. the gene of the described soybean WRKY class of claim 1 transcription factor of encoding.

3. gene according to claim 2 is characterized in that: the base sequence of this gene is shown in SEQ ID NO:2.

4. the plant expression vector that contains claim 2 or 3 described soybean WRKY class transcription factor genes.

5. the clone that contains claim 2 or 3 described soybean WRKY class transcription factor genes.

6. the host bacterium that contains claim 2 or 3 described soybean WRKY class transcription factor genes.

7. the application of the described soybean WRKY class of claim 1 transcription factor in the plant of cultivating the resistance raising.

8. claim 2 or the 3 described soybean WRKY class transcription factor genes application in the plant of cultivating the resistance raising.