CN102653556B - Transcription factor GmWRKY78 related to plant stress tolerance and its coding gene and application - Google Patents

Abstract

The invention discloses a plant stress tolerance-related transcription factor GmWRKY78, its coding gene and application. The protein provided by the present invention is the following (a) or (b): (a) a protein composed of the amino acid sequence shown in Sequence 1 in the Sequence Listing; (b) the amino acid sequence shown in Sequence 1 in the Sequence Listing after a A protein derived from Sequence 1 that has substitution and/or deletion and/or addition of several amino acid residues and is related to plant stress tolerance. The present invention is of great value for cultivating stress-tolerant plant varieties, especially for cultivating new varieties of crops and forest grasses resistant to abiotic stress (salt tolerance, drought tolerance and low temperature tolerance), and can be used for agriculture, animal husbandry and ecological environment management. The cultivation and identification of stress-tolerant plant varieties is of great significance for improving crop yield.

Description

Transcription factor GmWRKY78 related to plant stress tolerance and its coding gene and application

technical field

The invention relates to the field of biotechnology, in particular to a transcription factor GmWRKY78 related to plant stress tolerance and its coding gene and application.

Background technique

Changes in physical and chemical factors in the environment, such as drought, salinity, low temperature and other stress factors, have an important impact on the growth and development of plants. In severe cases, they will cause large-scale crop yield reduction. Cultivating stress-tolerant crops is one of the main goals of planting. At present, genetic engineering breeding has become one of the important methods to enhance crop stress tolerance. Higher plant cells have multiple pathways to respond to various stresses in the environment, and transcription factors play a role in regulating the expression of stress tolerance-related effector genes. Many types of transcription factors have been found in plants related to plant stress tolerance, for example: DREB in EREBP/AP2, bZIP, MYB, WRKY and so on.

The WRKY transcription factor is a plant-specific transcriptional regulator, and the cDNA of this family was originally cloned from sweet potato, wild oat, parsley and Arabidopsis. The name of this type of transcription factor comes from the fact that each protein contains 1-2 WRKY domains. The WRKY domain is a sequence of 60 amino acids, and the N-terminal contains the extremely conserved amino acid sequence WRKYGQK and a zinc finger domain. Almost all WRKY proteins have binding properties to W-box[(T)TGAC(C/T)], and can bind to W-box in their own promoters to participate in expression regulation. W-boxes exist in the promoters of many genes related to plant defense responses and mediate transcriptional responses induced by pathogen-derived elicitors. In Arabidopsis, the WRKY family has 74 members; in rice and soybean, there are more than 100 members. WRKY transcription factors are mainly related to plant disease resistance response, senescence, growth and development, and have been found to be related to stress tolerance since the 21st century. WRKY transcription factors that function under stress have been cloned from Arabidopsis, tobacco, potato and rice.

Soybean is the world's main source of edible vegetable oil, and provides high-protein feed for livestock and high-quality protein for humans. Their growth and yield are often affected by abiotic environmental stress. It is of great significance for soybean production to elucidate its stress tolerance mechanism and improve its stress tolerance.

Contents of the invention

One object of the present invention is to provide a plant stress tolerance-related transcription factor GmWRKY78 and its coding gene.

The protein related to plant stress tolerance provided by the present invention is called GmWRKY78, which is a transcription factor derived from soybean (Glycine max (L.) Merrill), and is as follows (a) or (b):

(a) a protein consisting of the amino acid sequence shown in Sequence 1 in the Sequence Listing;

(b) The amino acid sequence shown in Sequence 1 in the Sequence Listing is subjected to substitution and/or deletion and/or addition of one or several amino acid residues, and a protein derived from Sequence 1 that is related to plant stress tolerance.

Sequence 1 in the sequence listing consists of 306 amino acid residues and is a WRKY transcription factor in soybean.

The substitution and/or deletion and/or addition of one or several amino acid residues refers to the substitution and/or deletion and/or addition of no more than 10 amino acid residues.

In order to make the protein in (a) easy to purify, the amino-terminal or carboxy-terminal of the protein consisting of the amino acid sequence shown in Sequence 1 in the Sequence Listing can be linked with the tags shown in Table 1.

Table 1 Sequence of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

The substitution and/or deletion and/or addition in (b) above may be caused by natural variation or artificial mutagenesis.

The protein in (a) or (b) above can be synthesized artificially, or its coding gene can be synthesized first, and then biologically expressed. The protein-encoding gene in (b) above can be deleted by deleting one or several amino acid residue codons in the DNA sequence shown in Sequence 2 in the sequence listing, and/or performing one or several base pairs of missense mutation, and/or link the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end.

The gene GmWRKY78 encoding the protein GmWRKY also belongs to the protection scope of the present invention.

The gene can be a DNA molecule of (1) or (2) or (3) as follows:

(1) DNA molecules shown in sequence 2 in the sequence listing;

(2) A DNA molecule that hybridizes to the DNA sequence defined in (1) under stringent conditions and encodes a plant stress tolerance-related protein;

(3) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% of the DNA sequence defined in (1) , a DNA molecule having at least 98% or at least 99% homology and encoding a plant stress tolerance-related protein.

Described stringent condition can be as follows: 50 ℃, hybridize in the mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO ₄ and 1mM EDTA, wash in 50 ℃, 2×SSC, 0.1% SDS ; Can also be: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M NaPO ₄ and 1mM EDTA, rinse at 50°C, 1×SSC, 0.1% SDS; can also be: 50°C, in 7% Hybridize in a mixed solution of SDS, 0.5M NaPO ₄ and 1mM EDTA, rinse at 50°C in 0.5×SSC, 0.1% SDS; also: 50°C, in a mixture of 7% SDS, 0.5M NaPO ₄ and 1mM EDTA Hybridization in solution, rinse at 50°C, 0.1×SSC, 0.1% SDS; also: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M NaPO ₄ and 1mM EDTA, at 65°C, 0.1×SSC , 0.1% SDS; alternatively: in 6×SSC, 0.5% SDS solution, hybridize at 65°C, then wash the membrane once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS .

Sequence 2 in the sequence listing consists of 921 nucleotides, all of which are the coding sequence of the GmWRKY78 protein. The 1st to 3rd deoxyribonucleotides from the 5' end are the initiation codon ATG, and the 919th to 921st deoxyribose The nucleotide is the stop codon TAA.

Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the genes all belong to the protection scope of the present invention.

An existing plant expression vector can be used to construct a recombinant expression vector containing the gene. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment and the like. Such as pROKII, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Company), etc. The plant expression vector can also include the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopain synthase Nos gene), plant gene (such as soybean storage The untranslated region transcribed at the 3' end of protein gene) has similar functions. When using said gene to construct a recombinant plant expression vector, any enhanced promoter (such as cauliflower mosaic virus (CAMV) 35S promoter, corn ubiquitin promoter ( Ubiquitin)), constitutive promoters or tissue-specific expression promoters (such as seed-specific expression promoters), they can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct plant expression vectors , can also use enhancers, including translation enhancers or transcription enhancers, these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be in the same reading frame as the coding sequence to ensure that the entire sequence correct translation of . The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes (GUS gene, luciferase gene, etc.) Genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.) or chemical resistance marker genes (such as herbicide resistance genes). Such as the nptII gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinothricin, the hph gene that confers resistance to the antibiotic hygromycin, and the dhfr gene that confers resistance to metharexate gene, the EPSPS gene that confers resistance to glyphosate) the mannose-6-phosphate isomerase gene that confers the ability to metabolize mannose.

The recombinant vector is as follows 1) or 2):

1) insert the gene described in claim 2 or 3 into the recombinant vector obtained between the XbaI and KpnI recognition sites of the pROK II vector;

2) The recombinant vector obtained between the BamHI and KpnI recognition sites of the pBin438 vector by inserting the gene described in claim 2 or 3.

A pair of primers for amplifying the full length of the gene or any fragment thereof also falls within the protection scope of the present invention.

The primer pair can specifically be as follows (I) or (II):

(1) a primer pair consisting of DNA shown in sequence 3 of the sequence listing and DNA shown in sequence 4 of the sequence listing;

(II) A primer pair consisting of the DNA shown in Sequence 5 of the Sequence Listing and the DNA shown in Sequence 6 of the Sequence Listing.

The application of the protein or its coding gene in cultivating stress-tolerant plants is also within the protection scope of the present invention.

The application is that the gene is introduced into the target plant through the recombinant vector 1) in the recombinant vector to obtain the transgenic hairy root; the growth rate of the transgenic hairy root is greater than that of the empty vector hairy root The empty vector hairy root is the empty vector hairy root obtained by transferring pROK II into the target plant;

The stress tolerance is drought tolerance, salt tolerance and/or low temperature tolerance;

The plant is a dicotyledon or a monocotyledonous plant; the specific cotyledonous plant is soybean.

The second object of the present invention is to provide a method for breeding transgenic plants.

The method provided by the invention is to introduce the gene into the target plant to obtain a transgenic plant with higher stress tolerance than the target plant.

The gene is introduced into the target plant through the recombinant vector 2) in the above-mentioned recombinant vector;

The stress tolerance is drought tolerance;

The stress resistance is reflected in the elongation ratio of the main stem;

The target plant is a dicotyledon or a monocotyledonous plant; the specific dicotyledonous plant is Arabidopsis thaliana.

The transgenic plant is understood to include not only the first-generation transgenic plant obtained by transforming the target plant with the gene, but also its progeny. For transgenic plants, the gene can be propagated in that species, or transferred into other varieties of the same species, particularly including commercial varieties, using conventional breeding techniques. The introduction of the gene into the target plant will result in the synthesis of the protein in the target plant, and then the stress tolerance traits of the target plant will be improved.

The gene can be modified as follows first, and then introduced into the host to achieve better expression effect:

1) Modify and optimize according to actual needs, so that the gene can be expressed efficiently; for example, according to the codon preferred by the recipient plant, the codon can be changed while maintaining the amino acid encoded by the nucleotide sequence of the present invention to conform to Plant preference; in the optimization process, it is best to keep a certain GC content in the optimized coding sequence, so as to best realize the high-level expression of the introduced gene in the plant, wherein the GC content can be 35%, preferably more than 45%, more preferably greater than 50%, most preferably greater than about 60%;

2) modifying the gene sequence adjacent to the starting methionine to allow efficient initiation of translation; for example, using sequences known to be effective in plants for modification;

3) Linking with various plant-expressed promoters to facilitate its expression in plants; said promoters may include constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters ; the choice of promoter will vary with the temporal and spatial requirements of expression, and also depends on the target species; e.g. a tissue or organ-specific expression promoter, depending on what stage of development the recipient is desired; although proven source Many promoters for dicots are functional in monocots and vice versa, but ideally, dicot promoters are chosen for expression in dicots and monocot promoters are used for Expression in monocots;

4) Linking with suitable transcription terminators can also improve the expression efficiency of the gene of the present invention; for example, tml derived from CaMV, E9 derived from rbcS; any available terminators known to work in plants can be combined with The gene of the present invention is linked.

5) Introduce enhancer sequences, such as intron sequences (eg derived from Adhl and bronze) and viral leader sequences (eg derived from TMV, MCMV and AMV).

In practice, the gene of the present invention can also be targeted to cells. It can be realized by utilizing existing technologies in the art. For example, the target gene sequence derived from the target organelle is fused with the gene sequence of the present invention, and then introduced into the plant cell to achieve localization.

The expression vector carrying the gene can transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrical conduction, Agrobacterium-mediated, and transform the transformed The plant tissue is grown into a plant.

The experiment of the present invention proves that the expression of GmWRKY78 gene in soybean is induced by the treatment of high salt, drought, low temperature and plant hormone ABA. Afterwards, the GmWRKY78 gene was introduced into the root of Soybeaceae Feng No. 1 through the mediation of Agrobacterium rhizogenes, and the root system of GmWRKY78 gene was obtained. After salt and drought stress respectively, the growth state of the root system of GmWRKY78 gene was significantly better than that of The root system of the empty vector was transformed, indicating that the GmWRKY78 gene can significantly improve the salt tolerance of the plant. The present invention is of great value for cultivating stress-tolerant plant varieties, especially for cultivating new varieties such as crops and forest grasses resistant to abiotic stress (salt tolerance), and can be used for the development of stress-tolerant plant varieties required for agriculture, animal husbandry and ecological environment management. Cultivation and identification are of great significance to improving crop yield.

Description of drawings

Figure 1 is the quantitative PCR analysis of the expression characteristics of GmWRKY78 under various treatments

Figure 2 is a schematic diagram of the plant expression vector pROKII-GmWRKY78

Figure 3 is the establishment of the transgenic root system mediated by Agrobacterium rhizogenes

Figure 4 is the quantitative PC7 detection of the expression level of GmWRKY78 gene in the transgenic root system

Figure 5 is the drought tolerance identification of overexpressed GmWRKY78 roots

Fig. 6 is the salt tolerance identification of transgenic GmWRKY78 gene root system

Fig. 7 is the low temperature tolerance identification of transgenic GmWRKY78 gene root system

Figure 8 is the drought tolerance analysis of GmWRKY78 transgenic Arabidopsis

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

% in the following examples, unless otherwise specified, are mass percentages. In the quantitative experiments in the following examples, three repeated experiments were set up, and the data were the mean value or mean ± standard deviation of the three repeated experiments.

Glycine max L.Merr.Kefeng 1 (Glycine max L.Merr.Kefeng 1) was recorded in W.K.Zhang, Y.J.Wang, G.Z.Luo, J.S.Zhang, C.Y.He, X.L.Wu, J.Y.Gai, S.Y.Chen, QTL mapping of tenagronomic traits on the soybean( Glycine max L.Merr.) genetic map and theirassociation with EST markers, Theor.Appl.Genet, 2004, 108:1131-1139, the public can obtain from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences;

pROKII vector (binary expression vector) is described in D.C.Baulcombe, G.R.Saunders, M.W.Bevan, M.A.Mayo and B.D.Harrison, Expression of biologically active viral satelliteRNA from the nuclear genome of transformed plants.Nature 321(1986), pp.446-449 The public can obtain it from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences;

Agrobacterium rhizogenes K599 is recorded in Attila Kereszt, et al., Agrobacterium rhizogenes-mediated transformation of soybean to study of root biology. Nature Protocols, 2007, 2 (4), 549-552), the public can get from Professor Peter M Gressnon, The Obtained from the University of Queensland, St Lucia, Queensland 4072, Australia, or from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences with the consent of Prof. Peter M Gressnon (written consent).

The plant binary expression vector pBin438 is recorded in Li Taiyuan, Tian Yingchuan, Qin Xiaofeng, etc. Research on highly efficient insect-resistant transgenic tobacco [J]. Chinese Science (Series B), 1994, 24(3): 276-282, the public can download from China Institute of Genetics and Developmental Biology, Academy of Sciences.

Agrobacterium tumefaciens AGL1 was described in He Y, Jones HD, Chen S, Chen XM, Wang DW, Li KX, Wang DS, Xia LQ, Agrobacterium-mediated transformation of durum wheat(Triticumturgidum L.var.durum cv Stewart) with improved efficiency , J Exp Bot. 2010, 61(6): 1567-81, publicly available from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.

Wild-type Colombian ecotype Arabidopsis thaliana (Col-0) (hereinafter referred to as wild-type Arabidopsis) was purchased from Arabidopsis Biological Resource Center.

Example 1. Acquisition of soybean stress tolerance-related protein and its coding gene GmWRKY78

1. Discovery of the GmWRKY78 gene

The 3-week-old seedlings of Soybean Kefeng 1 were treated with 200mM NaCl for 1, 3, 6 and 12 hours respectively, and 1-2 grams of fresh leaves were taken from each treatment sample, ground in liquid nitrogen, and suspended in 4mol/L guanidine sulfide In the aqueous solution, the mixture was extracted with acidic phenol and chloroform, and absolute ethanol was added to the supernatant to precipitate to obtain total RNA (the expression level of the GmWRKY78 gene under salt stress was much higher than that under normal conditions, so the gene should be cloned, and the total RNA was obtained under salt stress. treatment of soybean seedlings). The extracted total RNA was reverse transcribed into cDNA. Using cDNA as a template, PCR amplification was performed with Primer-F and Primer-R to obtain PCR amplification products. The PCR amplification product was detected by 1% agarose gel electrophoresis, and a band with a molecular weight of about 900 bp was obtained, and the fragment was recovered with an agarose gel recovery kit (TIANGEN).

Primer-F: 5'-CCC TCTAGA ATGGATTATTCATCATGGATTA-3' (Xba I enzyme digestion recognition site is underlined) (SEQ ID NO: 3);

Primer-R: 5'-CCC GGTACC TTAATTATTTATTGTGCAACATTTTTC-3' (KpnI restriction recognition site is underlined) (SEQ ID NO: 4).

PCR amplification system (50 μl): cDNA 1 μl, 10×buffer 5 μl, dNTP (10 mM) 1 μl, Primer-F 1 μl, Primer-R 1 μl, Pfu DNA Polymerase (TaKaRa) 1 μl, ddH2O 40 μl. The amplification conditions were: 94°C for 3 minutes; 30 cycles of 94°C for 30s, 50°C for 30s, and 72°C for 30s; 72°C for 10m.

The recovered fragment (PCR product) was connected with pGEM-T Easy (Promega), the connected product was transformed into Escherichia coli DH5α competent cells, and positive clones were screened according to the carbenicillin resistance marker on the pGEM-T Easy vector to obtain the recovered Fragments of recombinant plasmids. Using the T7 and SP6 promoter sequences on the recombinant plasmid vector as primers to determine its nucleotide sequence, the sequencing results show that the PCR product has the nucleotides shown in sequence 2 in the sequence table, and the gene of the PCR product is named GmWRKY78, the coding region of the gene is 1-921 nucleotides from the 5' end of Sequence 2 in the sequence listing, and the protein encoded by the gene is named GmWRKY78, and the amino acid sequence of the protein is shown in the sequence 1 in the sequence listing. Sequence 2 in the sequence listing consists of 921 nucleotides, and sequence 1 in the sequence listing consists of 306 amino acid residues.

Sequence 2 in the Sequence Listing can also be artificially synthesized.

2. Expression characteristics of soybean GmWRKY78 gene under adversity stress treatment

The 3-week-old seedlings of Soybean Kefeng No. 1 were subjected to the following stress treatments:

1) Salt stress treatment (NaCl): move the seedlings into 200mM NaCl aqueous solution, room temperature, about 25°C;

2) Osmotic stress treatment (Dfought): The roots of the seedlings were carefully absorbed to remove water, placed on filter paper and exposed to air at room temperature (25°C);

3) Low temperature stress treatment (Cold): immerse the seedlings in pre-cooled water and place them in a refrigerator at 4°C;

4) Abscisic acid (ABA) treatment, immerse the seedling roots in 100 μM ABA aqueous solution at room temperature, about 25°C.

5) CK treatment: Seedlings without any treatment are recorded as CK;

6) Water treatment: immerse the seedlings in distilled water, and the treatment temperature is about 25°C;

Collect 1 g of fresh leaves at 1, 3, 6, and 12 hours for each treatment, grind them in liquid nitrogen (collect the leaves directly in the CK group), suspend them in 4 mol/L guanidine thiohydrogen aqueous solution, and extract the mixture with acidic phenol and chloroform , adding absolute ethanol to the supernatant to obtain total RNA. Quantitative PCR analysis was performed on the expression characteristics of GmWRKY78 gene under the above treatments, and the primers were Primer-F and Primer-R. The experiment was repeated three times, and the results were average ± standard deviation.

The result is shown in Figure 1,

The relative expression level of GmWRKY78 gene treated with CK was 0.03±0.005;

The relative expression of GmWRKY78 gene in water treatment 1, 3, 6, 12h was 0.03±0.002, 0.02±0.001, 0.02±0.001, 0.02±0.001;

The relative expression levels of GmWRKY78 gene were 0.07±0.015, 0.075±0.015, 0.15±0.04, 0.17±0.05 at 1, 3, 6 and 12h of osmotic stress treatment;

The relative expression levels of GmWRKY78 gene were 0.025±0.002, 0.11±0.03, 0.23±0.09, 0.34±0.08 at 1, 3, 6 and 12h of low temperature stress treatment;

The relative expression levels of GmWRKY78 gene were 0.11±0.03, 0.26±0.10, 0.095±0.03, 0.24±0.07 at 1, 3, 6 and 12h of salt stress treatment;

Abscisic acid (ABA) treatment 1, 3, 6, 12h relative expression of GmWRKY78 gene was 0.1±0.06, 0.04±0.005, 0.01±0.001, 0.008±0.001;

It can be seen from the figure that the GmWRKY78 gene responds to low temperature, drought, high salt and ABA treatment, and its expression is obviously induced by low temperature, drought and high salt treatment except for ABA treatment. It increased sharply at 1 hour, then decreased, and dropped to the control level by 6 hours. GmWRKY78 expression did not change significantly when water was treated, and it was confirmed that the expression of GmWRKY78 gene was induced by abiotic stress.

Embodiment 2, the overexpression of GmWRKY78 gene can improve the stress tolerance of recipient plant

1. Establishment of Agrobacterium rhizogenes-mediated transgenic root system method

The Agrobacterium rhizogenes infection method was carried out according to the method of Attila Kereszt et al. (Attila Kereszt, et al., Agrobacterium rhizogenes-mediaded transformation of soybean to study of root biology, Nature Protocols, 2007, 2(4), 549-552).

The specific operation is as follows:

1) First, pBI121 containing the GUS gene (Datla RS, Hammerlindl JK, Panchuk B, PelcherLE, Keller W, Modified binary plant transformation vectors with the wild-typegene encoding NPTII, Gene.1992Dec 15; 122(2): 383-4 , the public can obtain from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences), and introduced into Agrobacterium rhizogenes K599 by electric shock method to obtain recombinant Agrobacterium. Extract the plasmid of recombinant Agrobacterium, and use PCR method to identify the target gene contained in the recombinant Agrobacterium. The primers used are forward: 5'-ATGTTACGTCCTGTAGAAAC-3'reverse: 5'-AGCGGGTAGATATCACACTC-3'

The amplified fragment is the 5' end 800bP segment of the GUS gene, which proves that the plasmid is pBI121, which contains the GUS gene, and the recombinant Agrobacterium containing the plasmid is a positive recombinant Agrobacterium, named K599/pBI121.

2) Seeds of Soybean Kefeng No. 1 were sown in vermiculite, and waited to grow 2 true leaves for later use.

3) Streak activation of K599/pBI121 in LB solid medium containing 50 mg/L kanamycin, pick a single colony and shake overnight in LB liquid medium containing 50 mg/L kanamycin to obtain K599/pBI121 Agrobacterium rhizogenes.

4) Use the Agrobacterium rhizogenes bacteria liquid obtained in step 3) to inoculate with a syringe the soybean seedlings obtained in step 2) containing two true leaves for 6 days (see Figure 3A, 6 days seedling age, at a distance of 1-2 from the vermiculite surface. Inject at 2cm.), moisturizing growth: light for 16 hours, temperature 25°C, humidity 50%. After 2 weeks, hairy roots grow (Fig. 3B), which can be used for transgene identification and stress experiments.

When the length of the hairy root is 5-10 cm, the old root is cut off, and the new hairy root is the transformed root, which can be further used for transgenic identification.

Four new hairy roots were subjected to GUS staining, and the hairy roots were directly placed in the following staining solution: 50mM NaP04, pH7.2, 2mM X-gluc, 0.5mM K3Fe(CN)6, and 0.5mM K4Fe(CN)6 In 37°C overnight, the hairy roots were washed (decolorized) with a series of ethanol concentrations of 70, 50, and 30%, and then placed in distilled water for observation. The results are shown in Figure 3C. It can be seen that GUS staining indicates that exogenous The gene GUS has been expressed in newly grown hairy roots, indicating that the above method can transform exogenous genes into hairy roots (containing pBI121). The above-mentioned new hairy roots are transgenic hairy roots.

Because the pBI121 vector contains the GUS gene, when establishing the hairy root transformation system, the expression of the GUS gene is blue and easy to detect, so the pBI121 vector is used. In the formal experiment, it is better to use NPTII labeling, so it is replaced by pROK II vector.

2. Construction of recombinant expression vector pROK II-GmWRKY78

Use restriction endonuclease XbaI and KpnI to double digest the PCR product obtained by embodiment 1Primer-F and Primer-R as primers, reclaim the digested product, and obtain the digested product with the plant expression vector pROKII through the same restriction enzyme digestion The vector backbone is ligated to obtain the ligated product. The ligation product was transformed into Escherichia coli to obtain a transformant. Extract the plasmid of the transformant and send it for sequencing. This plasmid is the carrier obtained by inserting the sequence 2 in the sequence listing between the XbaI and KpnI restriction sites of pROKII. The vector is named pROKII-GmWRKY78, and in the sequence listing The sequence 2 of is located behind the CaMV 35S promoter. The schematic diagram of the structure of the recombinant expression vector pROKII-GmWRKY78 is shown in FIG. 2 .

3. Obtaining root system with overexpression of GmWRKY78 gene

1) The recombinant expression vector pROK II-GmWRKY78 obtained in the above 2 was introduced and transformed into Agrobacterium rhizogenes K599 by electric shock method to obtain recombinant Agrobacterium.

The plasmid of the recombinant Agrobacterium was extracted and sent for sequencing. The result was that the plasmid was pROKII-GmWRKY78, and the recombinant Agrobacterium containing the plasmid was named K599/pROKII-GmWRKY78.

2) Use a syringe to inoculate the recombinant Agrobacterium K599/pROK II-GmWRKY78 into soybean Kefeng No. 1 seedlings that have grown for 6 days and contain two true leaves. For the specific method, see step 4) of the above 1, Figure 3A, moisture growth: 16 hours of light , temperature 25°C, humidity 50%. After 2 weeks, hairy roots grow and become transformed hairy roots. A total of 26 transgenic GmWRKY78 hairy root systems were obtained, which can be further used for transgenic identification and stress tolerance testing.

In the same way, the empty vector pROK II was transferred into soybean Kefeng 1 seedlings, and 26 hairy roots transformed with the empty vector were obtained as experimental controls.

3) Total RNA was extracted from hairy roots transfected with GmWRKY78 and hairy roots transformed with empty vector, and reverse transcribed into cDNA. Using cDNA as a template, the expression of GmWRKY78 gene was analyzed with Primer-78F and Primer-78R. The Real-Time PCR reaction uses TOYOBO's RealTime PCR Master Mix kit and operates according to the instructions. Primer-78F and Primer-78R were used to detect the expression level of GmWRKY78 gene; the soybean GmTubulin gene was used as internal standard, and the primers used were Primer-TF and Primer-TR. The experiment was repeated three times, and the results were average ± standard deviation.

Primer-78F: 5'-GTAACAACAGGTTCCAACCGTTC-3' (SEQ ID NO: 5)

Primer-78R: 5'-CTTCTGGTGATTCAGTTTTGGGATT-3' (SEQ ID NO: 6)

Primer-TF: 5'-AACTCCATTTCGTCCATTCCTTC-3'

Primer-TR: 5'-TTGAGTGGATTCCCAACAACG-3'

The results are shown in Figure 4, wherein, pROKII-GmWRKY78 is the hairy root transformed with GmWRKY78, and pROKII is the hairy root transformed with empty vector. It can be seen from the figure that the relative expression level of GmWRKY78 in the hairy root transformed with GmWRKY78 is 4.25±0.2; The relative expression level of GmWRKY78 detected in the hairy roots of the empty vector was the expression of the original GmWRKY78 in soybean, which was 0.75±0.1; quantity.

4. Identification of drought tolerance, salt tolerance and low temperature tolerance of transgenic GmWRKY78 root system

The experimental samples were the root system of empty vector and the root system of GmWRKY78.

There were 2 untreated root systems of the two transgenic plants.

1) Drought tolerance identification:

The hairy roots of the transgenic GmWRKY78 gene and the transgenic vector hairy roots were respectively immersed in 1% (volume percentage) and 2.5% PEG (polyethylene glycol 6000) for 3 days at 25°C to transform the hairy roots of GmWRKY78 and the transgenic Empty vector hairy roots were grown in water as a control. There were 8 root systems treated with two concentrations of PEG. The experiment was repeated three times, and the results were averaged.

After 3 days of treatment, take photos and observe, the results are shown in Figure 5A. It can be seen that, during water treatment, there was no significant difference in the growth of the hairy roots of the transgenic GmWRKY78 gene (GmWRKY78) and the transgenic vector hairy roots (pROKII). After treatment with 1% PEG and 2.5% PEG, there was a significant difference in the growth rate of hairy roots between transgenic GmWRKY78 and transgenic vector hairy roots.

Specifically measure each group of root systems, first calculate the growth rate of each hairy root=(root length after processing-handling front root length)/handling front root length, then get mean ± standard deviation;

The growth rate is plotted as shown in Figure 5B. When the PEG concentration is 1%, the growth rate of the hairy root of the empty vector (pROKII) is about 48 ± 7%, while the growth rate of the hairy root of the GmWRKY78 (GmWRKY78) is about 70±8%. The growth rate of the hairy root of the transgenic GmWRKY78 gene was slightly different from that of the empty vector. When the concentration of PEG increased to 2.5%, the growth rate of the hairy root of the empty vector decreased to 25%, while that of the transgenic GmWRKY78 hairy root decreased to 30%, which was still slightly higher than that of the control.

2) Identification of salt tolerance:

Ten of the hairy roots of transgenic GmWRKY78 and transgenic vectors were treated with 100mM NaCl aqueous solution for 3 days, then immersed in 100mM NaCl solution at 25°C. The experiment was repeated three times, and the results were average ± standard deviation.

After 3 days of treatment, take pictures and observe the results, as shown in Figure 6A, the phenotypes of the hairy roots of the empty vector (pROK II) and the hairy roots of GmWRKY78 (GmWRKY78) that were treated with 100mM NaCl for 3 days, as can be seen, water treatment (Normal condition) There is no significant difference between the hairy roots of the empty vector and the hairy roots of the GmWRKY78, but there is a significant difference between the two under the treatment of 100mM NaCl.

The measurements are as follows:

The formula for calculating the growth rate of hairy root length is the same as above.

The growth rate is plotted as shown in Figure 6B, the length growth rate of the transgenic hairy root of GmWRKY78 gene (please check whether the hairy root or the root system is suitable) is 23 ± 8% after being treated with 100mM NaCl aqueous solution. However, the growth rate of the hairy root length of the empty carrier treated by 100mM NaCl aqueous solution was 7±6%, and there was a significant difference between the two.

3) Identification of low temperature resistance:

Take 10 transgenic GmWRKY78 gene root systems and 10 transgenic root systems and treat them at 4°C for 3 days. The experiment was repeated three times, and the results were averaged.

After 3 days of treatment, take pictures and observe the results, as shown in Figure 7A, the phenotypes of the hairy roots of the empty vector (pROKII) and the hairy roots of GmWRKY78 (GmWRKY78) treated at 4°C for 3 days, it can be seen that the water treatment ( Under normal conditions) there was no significant difference between the empty vector and the GmWRKY78-transferred root system, but there was a significant difference between the two under the 4°C treatment.

The measurements are as follows:

The formula for calculating the growth rate of hairy root length is the same as above.

The growth rate is plotted in Figure 7B, the growth rate of hairy roots transformed into GmWRKY78 treated at 4°C was 7.5±2%, and the growth rate of hairy roots transformed into empty vector was 0 after treated at 4°C. There is a very significant difference between the two.

The identification of the stress tolerance of the above-mentioned empty vector hairy root and the transgenic GmWRKY78 hairy root showed that GmWRKY78 was related to plant tolerance to abiotic stress, and the overexpression of GmWRKY78 in soybean increased the salt tolerance, drought tolerance and low temperature tolerance of transgenic soybean . The GmWRKY78 gene can be used as the target gene of genetic engineering for improving plant resistance to abiotic stress.

Example 3, Functional identification of transgenic GmWRKY78 Arabidopsis

1. Recombinant vector pBin438-GmWRKY78

Using the cDNA obtained in Example 1 (salt treatment is required because the expression level of the target gene increases after salt treatment) as a template, primer 1 and Primer-R were used as primers for PCR amplification.

Primer 1: 5'-CCCTTTCGGATGGATTATTCATCATGGATTA-3' (SEQ ID NO: 7)

Primer-R: 5'-CCCGGTACCTTAATTTATTATTGTGCAACATTTTTC-3' (SEQ ID NO: 4)

Digest the above PCR product with BamHI and KpnI, recover it, and ligate it with the large fragment of the vector obtained from the plant binary expression vector pBin438 after the same digestion to obtain the ligated product, transfer it into Escherichia coli, obtain a transformant, and extract the transformant The plasmid was sent for sequencing. This plasmid is the vector obtained by inserting sequence 2 in the sequence table between the BamHI and KpnI restriction sites behind the CaMV35S promoter of pBin438. The plasmid is named pBin438-GmWRKY78, which is recombinant Vector, the structure diagram of the recombinant vector is shown in Figure 8A.

2. Recombinant Agrobacterium tumefaciens AGL1/pBin438-GmWRKY78

The recombinant vector pBin438-GmWRKY78 was introduced into Agrobacterium tumefaciens AGL1 by electric shock method to obtain recombinant bacteria. PCR identification, with primer 1 and Primer-R as primers, a fragment of about 900bp was obtained, so it was a positive recombinant bacterium. The plasmid was extracted from the positive recombinant bacterium and sent for sequencing. The result was that the plasmid was pBin438-GmWRKY78, which further proved that the bacterium was The positive strain was named AGL1/pBin438-GmWRKY78.

3. Obtaining and identification of transgenic Arabidopsis thaliana

The recombinant Agrobacterium AGL1/pBin438- GmWRKY78 was transferred into Colombian ecotype Arabidopsis thaliana (Col-0), and the specific operation was as follows: the flower of wild-type Arabidopsis Col-0 was soaked in the transformation solution (MS 0.011g, Sucrose 0.25g, Silwet 0.5μl , diluted to 5ml with distilled water, pH 5.8) for 10 seconds, took it out and put it into MS medium for dark cultivation for 8 hours to obtain T ₀ generation transformed seeds, which were sown in the seed containing kanamycin (50mg/L ) on the MS medium, and the T ₀ generation was transferred to GmWRKY78 Arabidopsis. Harvest seeds T ₁ generation seeds, sow on the MS selection medium containing kanamycin (50mg/L), obtain 18 strains T ₁ generation transgenic GmWRKY78 Arabidopsis thaliana. When the T ₁ generation transgenic GmWRKY78 Arabidopsis grows to 4-6 leaves, move them to vermiculite for growth.

According to the above method, the empty vector pBin438 was transferred into the wild-type Colombian ecotype Arabidopsis thaliana (Arabidopsisthaliana) (Col-0), and 10 strains of T ₀ generation empty vector Arabidopsis were obtained. After PCR identification, the primers were primer 1 and Primer -R, the target fragment was not obtained, and the T ₀ generation empty vector Arabidopsis was obtained.

The total RNA of wild-type Arabidopsis thaliana and _T1 generation transgenic Arabidopsis GmWRKY78 was extracted for RT-PCR analysis, and the primers used were primer 1 and Primer-R.

The results showed that 10 of the 18 Arabidopsis thaliana transgenic to GmWRKY78 in the T ₀ generation expressed the GmWRKY78 gene. The seeds of the plants with positive molecular identification were harvested, and the seeds of each individual plant were sown separately, and the screening was continued with kanamycin to observe the segregation of the _T1 generation, and so on until the _T3 generation obtained genetically stable transgenic lines. A total of 10 stably inherited Arabidopsis lines of the T ₃ generation transgenic to GmWRKY78 were obtained, numbered 78-1, 7-11 and 78-24.

Harvest seeds from the single plant of Arabidopsis thaliana transformed into the empty vector in the T ₀ generation, sow the seeds of each individual plant separately, and continue to screen with kanamycin to observe the segregation of the T ₁ generation, and repeat until the T ₃ generation is transformed into the empty vector Arabidopsis thaliana .

T ₁ generation represents the seeds produced by T ₀ generation self-crossing and the plants grown from it, T ₂ generation represents the seeds produced by T ₁ generation self-crossing and the plants grown from it, T ₃ generation represents the T ₂ generation self-crossing The seed produced by intercourse and the plant that grows from it.

Extract the RNA of 10 stably inherited T ₃ generation Arabidopsis lines transformed into GmWRKY78 thaliana, and reverse transcribe to obtain cDNA as a template. The primers used are primer ₁ and Primer-R. Arabidopsis was used as the control, and the results are shown in Figure 8B, wherein, cDNA is the cDNA of wild-type Arabidopsis, CK is without any template, and W78-1, W78-11 and W78-24 are the target gene expression screened respectively. The three high T ₃ generations were transferred to the GmWRKY78 Arabidopsis line, and it can be seen that the three lines are positive lines.

There was no significant difference in the results between wild type Arabidopsis and _T3 generation empty vector Arabidopsis.

4. Functional identification of transgenic Arabidopsis thaliana

The 3-week-old seedlings of three T ₃ generation transgenic GmWRKY78 Arabidopsis lines (W78-1, W78-11 and W78-24) were placed under continuous light at 26-28°C, relative humidity 15-20%, and 300mM mannitol was treated for 14 days, and then the phenotype was observed and the length of the main stem was counted. The wild-type Arabidopsis and the _T3 generation empty vector Arabidopsis were used as controls, and the above plants were also not treated with 300mM mannitol. The experiment was repeated three times in total, and in each repetition, 15 plants of each strain were tested.

Photographic observation is shown in Figure 8C. The upper picture of 8C shows the phenotypes of the transgenic lines after 14 days of treatment with 300mM mannitol, and the lower picture shows the untreated control; it can be seen that the untreated wild-type control and the transgenic plants There was no significant difference in phenotype, while the growth of the control treated with mannitol for 14 days was significantly inhibited, while the growth of the three transgenic lines was significantly better than that of the control.

The results are shown in Figure 8D, which is the statistics of the main stem elongation ratio of the control and transgenic plants after the above-mentioned mannitol treatment, wherein, CK is wild-type Arabidopsis, and W78-1, W78-11 and W78-24 are 3 Three T ₃ generations were transferred to the GmWRKY78 Arabidopsis line;

CK main stem elongation ratio (main stem length after treatment-main stem length before treatment)/main stem length before treatment) is 0.5;

The main stem elongation ratio of W78-1 is 1.3;

The main stem elongation ratio of W78-11 is 1.5;

The main stem elongation ratio of W78-24 is 1.4;

It can be seen from the above that the elongation ratio of the main stem of the transgenic lines 78-1, 78-11 and 78-24 is better than that of the wild type Arabidopsis.

The above experiments not only show that the overexpression of GmWRKY78 improves the drought tolerance of transgenic plants, but also show that the transgenic soybean root system can be used as a detection system for the correlation between target genes and stress tolerance. The stress tolerance of transgenic soybean roots can illustrate the correlation between the target gene and plant stress tolerance.

Claims (10)

1. protein, the protein of being formed by the aminoacid sequence shown in the sequence in the sequence table 1.

2. the gene of coding claim 1 described albumen.

3. gene as claimed in claim 2, it is characterized in that: described gene is the dna molecular of following (1) or (2):

(1) dna molecular shown in the sequence 2 in the sequence table;

(2) the dna sequence dna hybridization that under stringent condition, limits with (1) and the dna molecular of coded plant stress tolerance correlative protein.

4. the recombinant vectors that contains claim 2 or 3 described genes.

5. recombinant vectors as claimed in claim 4 is characterized in that:

Described recombinant vectors is following 1) or 2):

1) claim 2 or 3 described genes are inserted the recombinant vectors that the multiple clone site of pROK II carriers obtains;

2) claim 2 or 3 described genes are inserted the recombinant vectors that the multiple clone site of pBin438 carriers obtains.

6. contain claim 2 or 3 described expression of gene boxes.

7. the transgenic cell line that contains claim 2 or 3 described genes.

8. the reorganization bacterium that contains claim 2 or 3 described genes.

9. the described albumen of claim 1 or its encoding gene application in cultivating plant with adverse resistance;

Described resistance of reverse is drought tolerance, salt tolerance and/or low temperature resistant;

Described plant is Arabidopis thaliana or soybean.

10. a method of cultivating transgenic plant is that claim 2 or 3 described genes are imported in the purpose plant, obtains the transgenic plant that resistance of reverse is higher than described purpose plant;

Claim 2 or 3 described genes are by the described recombinant vectors 2 in the described recombinant vectors of claim 5) import in the described purpose plant;

Described resistance of reverse is drought tolerance; Described resistance of reverse is embodied in stem elongation ratio;

Described purpose plant is Arabidopis thaliana or soybean.

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