CN117683104A - Soybean drought resistance gene GmACO9, protein encoded by same and application thereof - Google Patents

Abstract

The invention provides a soybean drought resistance gene GmACO9 and a protein coded by the same and application thereof, which comprise the steps of transforming the GmACO9 gene or a vector or host cell containing the GmACO9 gene into soybean plant cells or tissues and culturing to obtain a soybean plant with improved drought resistance. Meanwhile, the invention discloses application of the GmACO9 gene or a vector or host cell containing the GmACO9 gene in cultivation of drought-resistant soybeans. The invention has great theoretical and application value for soybean breeding and related application research.

Description

Soybean drought resistance gene GmACO9, protein encoded by same and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a soybean drought resistance gene, a protein encoded by the soybean drought resistance gene and application of the soybean drought resistance gene.

Background

Drought severely affects the growth and development of plants, and in order to cope with drought plants, a series of response patterns have been developed, in which phytohormones play an important role in drought tolerance in plants (Fang et al, 2015). Ethylene is one of the well-known 'adversity hormones', and under normal conditions, the ethylene content is low in plants, and the ethylene content is rapidly increased under adversity stress, so that the ethylene participates in regulating and controlling abiotic stress such as drought, salt, heat, waterlogging and the like of the plants. And since ethylene is a gas, there is no need to transport from the synthesis site to the action site, and thus the synthesis of ethylene is an important factor affecting the content of ethylene in the body. The biosynthetic pathway of ethylene has been well established to date. Methionine is first converted to SAM by S-adenosylmethionine Synthase (SAM) and then SAM is reacted by 1-aminocyclopropyl-1-carboxylic Acid (ACC) synthase (ACS) to form ACC and 5-methylthioadenosine, ACS being a member of the pyridoxal 5-phosphate dependent transaminase family. The ACC produced oxidizes to ethylene under the action of ACC oxidase (ACO) (Bakshi et al 2015). ACS is the rate-limiting enzyme in ethylene synthesis, but there has been increasing evidence in recent years that ACO plays an important role in ethylene synthesis (Shi et al, 2006).

ACO belongs to family members of the family subfamily DOXC of plant 2-ketoglutarate and ferrous ion dependent oxidases (2-Oxoglutarate and Fe (II) -dependent dioxygenase,2 OGD), amino acids histidine 177 (H177) and aspartic acid 179 (D179) are amino acids essential for the catalytic site of ACO, H177 being the binding site for the ferrous ion of the catalyst (Houben and Van de Poel, 2019). An arabidopsis thaliana NAC transcription factor family member SPEEDY HYPONASTIC GROWTH (SHYG) gene can bind to and regulate the expression of ACO5 on the ACO5 promoter to affect ethylene synthesis (Rauf et al, 2013). CsWIP1 can bind to the CsACO2 promoter in cucumber to inhibit its expression, thereby affecting ethylene synthesis to regulate plant sex (Chen et al, 2016). The TuACO3 gene expression of wheat is induced by powdery mildew treatment, and over-expression of the gene in wheat can improve the resistance of the wheat to powdery mildew, promote ethylene synthesis, reduce the synthesis amount of ethylene by reducing the expression of the gene, so that the sensitivity of transgenic plants to powdery mildew is enhanced (Zheng et al 2020). There are 16 ACO genes in soybean, and research on the functions and regulation mechanisms of these genes has very important biological significance for understanding the effect of ethylene synthesis in soybean plants on drought resistance of soybean.

Disclosure of Invention

The invention aims to provide an application of a gene for encoding 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) in drought resistance of soybeans.

The invention analyzes genetic control sites of soybean standard drought resistance index on the whole genome level by a correlation analysis method, and clones a related gene Glyma.08G029200 (also called GmACO9 or GmACO9 in the text) for regulating the drought resistance of soybean by integrating haplotype, gene expression profile and homologous gene function annotation and applying means of molecular biology and comparative genomics ^Hap1 、GmACO9 ^Hap2 、GmACO9 ^Hap3 And GmACO9 ^Hap4 ) The invention discovers the function of the gene in regulating and controlling the drought resistance of soybean for the first time, and provides theoretical basis and gene resources for subsequent molecular assisted breeding and molecular design breeding.

In the specific embodiment of the invention, the cDNA sequence of the GmACO9 gene is shown as SEQ ID NO.5, SEQ ID NO. 6, SEQ ID NO. 7 or SEQ ID NO. 8; preferably, the GmACO9 ^Hap3 The cDNA sequence of the gene is shown as SEQ ID NO. 7. In a specific embodiment of the invention, the genomic DNA (gDNA) sequence of the GmACO9 gene is shown in SEQ ID NO. 9-SEQ ID NO. 12.

In the present invention, a recombinant vector containing a target gene can be constructed using an existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like. To facilitate identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example by adding genes encoding enzymes or luminescent compounds which produce a color change, antibiotic markers or chemical resistance marker genes which are expressed in the plants, etc. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene. Enhancers may also be included in the plant expression vector to increase the expression of the inserted nucleotide fragment.

In order to achieve the above object, the present invention also provides a method for obtaining transgenic soybean, which comprises introducing the aforementioned nucleic acid or vector or host cell comprising the aforementioned gene into a soybean of interest to obtain a transgenic soybean exhibiting improved drought resistance as compared with the soybean of interest.

Among them, the method of introducing the soybean of interest may be by transforming plant cells or tissues using conventional biological methods such as Ti plasmid, ri plasmid, plant viral vector, direct DNA transformation, microinjection, electric conduction, agrobacterium mediation, etc., and culturing the transformed soybean cells or tissues into plants.

In order to achieve the above object, the present invention also provides the use of a protein as defined above or a nucleic acid as defined above or a vector or host cell comprising the above gene in soybean genetic engineering.

Wherein, the soybean genetic engineering is preferably soybean genetic engineering aiming at improving drought resistance of soybean.

The functions of the soybean drought resistance related genes and the encoded proteins thereof in the aspect of regulating and controlling the soybean drought resistance are discovered for the first time by the applicant, and the phenotype analysis verification of transgenic plants and wild plants shows that the expression of the soybean drought resistance related proteins can improve the drought resistance of transgenic soybeans. The invention has great theoretical and application value for soybean drought resistance breeding and related application research.

In summary, the present invention provides the following embodiments:

1. the amino acid sequence of the drought-resistant protein is shown as SEQ ID NO.1, SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4.

2. A polynucleotide sequence encoding the drought resistance protein of claim 1.

3. The polynucleotide sequence of item 2 which is GmACO9 ^Hap1 Gene GmACO9 ^Hap2 Gene, gmACO9 ^Hap3 Gene or GmACO9 ^Hap4 A gene, wherein the GmACO9 ^Hap1 Gene, gmACO9 ^Hap2 Gene, gmACO9 ^Hap3 Gene or GmACO9 ^Hap4 The cDNA sequence of the gene is shown as SEQ ID NO.5, SEQ ID NO. 6, SEQ ID NO. 7 or SEQ ID NO.8 respectively.

4. The polynucleotide sequence of item 3, wherein the GmACO9 ^Hap1 Gene, gmACO9 ^Hap2 Gene, gmACO9 ^Hap3 Gene or GmACO9 ^Hap4 The gDNA sequence of the gene is shown as SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12 respectively.

5. A vector, wherein the vector comprises the polynucleotide sequence of any one of items 2-4, wherein the vector is a plant expression vector, preferably a binary agrobacterium vector.

6. A host cell comprising the polynucleotide sequence of any one of clauses 2-4 or the vector of clause 5, wherein the host cell is selected from the group consisting of an Escherichia coli cell, an agrobacterium Agrobacterium tumefaciens cell, or a plant cell.

7. The drought-resistance protein of item 1, the use of the polynucleotide sequence of any one of items 2 to 4 for breeding plants, preferably leguminous plants, more preferably soybean, with increased drought resistance.

8. A method of growing a transgenic plant with increased drought resistance comprising introducing the drought resistance protein of item 1, the polynucleotide sequence of any one of items 2-4, or the vector of item 5 or the host cell of item 6 into a plant cell or tissue of interest to obtain a transgenic plant, said transgenic plant having increased drought resistance compared to said plant of interest, said plant being a leguminous plant, preferably soybean.

Drawings

FIG. 1 shows cloning of the GmACO9 gene. And a, carrying out whole-gene association analysis on the soybean standard drought resistance index character, wherein the threshold value is 5.2. And b, QQ dot diagram of standard drought resistance index character. c, linkage disequilibrium display of SNPs in No.8 chromosome standard drought resistance index candidate intervalShown. And d, analyzing the expression quantity of the gene screened by the haplotype analysis in the candidate interval after the treatment of 15% PEG solution. e, four major haplotype structures of GmACO9 (left) and phenotype value distribution of the four haplotypes in the natural population (right), wherein GmACO9 ^Hap3 Is a dominant haplotype. GmACO9 gene expression is induced by PEG stress treatment. g GmACO9 ^Hap1 And GmACO9 ^Hap3 Comparison of the enzymatic activities of the two ACOs.

FIG. 2 shows a GmACO9 gene prokaryotic expression vector pMal-c2x-GmACO9 ^Hap1 And pMal-c2x-GmACO9 ^Hap3 Is a schematic diagram of the map.

FIG. 3 shows GmACO9 gene overexpression vector pTF101.1-35s: gmACO9 ^Hap3 And RNAi transformation vector pFGC5941-GmACO 9.

FIG. 4 shows the wild type DN50 (Dongnong 50) and transfected pTF101.1-35s: gmACO9 ^Hap3 Plasmid-derived overexpressing transgenic plants (OE 1 ^GmACO9 And OE2 ^GmACO9 ) And RNAi transgenic plants (RNAi-1) obtained by transfection of the pFGC5941-GmACO9 plasmid ^GmACO9 And RNAi-2 ^GmACO9 ) Phenotype comparison of (3). Wherein a: DN50 wild type, OE1/2 ^GmACO9 And RNAi-1/2 ^GmACO9 Phenotype comparison under normal growth conditions. DN50 wild-type, OE1/2 ^GmACO9 And RNAi-1/2 ^GmACO9 Phenotype comparison after water-break treatment. And c, relative expression quantity of GmACO9 in the over-expression transgenic plant and RNAi transgenic plant. d-e: DN50 wild type, OE1/2 ^GmACO9 And RNAi-1/2 ^GmACO9 And (5) carrying out rehydration survival statistics on transgenic plants after 14 days of water-break treatment.

Detailed Description

The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.

The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, were either conventional or selected according to the commercial specifications. The test materials used in the examples described below, unless otherwise specified, are all commercially available conventional biochemical reagents. The quantitative tests in the following examples were all set up in triplicate and the results averaged.

In the following examples, the transformed recipient is dongnong 50 (DN 50), where DN50 is the black longjiang province variety (black bean 2007022). pTF101.1 vector, pFGC5941 vector and Agrobacterium strains EHA101, EHA105 were purchased from China plasmid vector strain cell gene collection (Biovector Science Lab, inc).

Consumables such as the enzyme digestion recovery kit are purchased from New England Biolabs and Tiangen Biochemical technologies (Beijing) limited.

Example 1 discovery of GmACO9 protein and its coding Gene

The inventors performed a whole genome association analysis (GWAS) using drought resistance index of 584 parts of soybean germplasm material. The calculation of Principal Component Analysis (PCA) and of the Kinship coefficient (Kinship) was carried out using the SNP with the minimum allele frequency (Minor allele frequency, MAF). Gtoreq.0.03. Wherein, the first two components of the principal component analysis are selected for controlling the group structure, and in addition, the genetic relationship is reflected by using a matrix of the sizing-Nichols (BN) generated by EMMA. Using the software EMMAX (Kang et al, 2010) for whole genome association analysis, we detected a signal on chromosome 16 and chromosome 8 that was significantly associated with the standard drought resistance index (see fig. 1a, b). In the present invention, only the segment located in the number 8 stain was analyzed, and in combination with transcriptome data, haplotype analysis and homologous gene function annotation, a soybean drought resistance-related protein was found in this segment (see FIGS. 1c, d). It is named GmACO9 protein (GmACO 9), the amino acid sequence of the protein is shown in any one of SEQ ID NO.1 to SEQ ID NO.4, and the gene encoding GmACO9 protein is named GmACO9 (or GmACO 9) ^Hap1 、GmACO9 ^Hap2 、GmACO9 ^Hap3 And GmACO9 ^Hap4 ) The cDNA sequences of the genes are shown in SEQ ID NO.5 to SEQ ID NO.8 respectively.

The invention further provides a nucleotide polymorphism of the SNP locus of the GmACO9 gene in the soybean genome, as shown in FIG. 1e, which can be used for detecting soybean variety background. For example, introducing haplotype 3 (Hap 3) of GmACO9 (i.e., the dominant haplotype of the GmACO9 gene) into other haplotype soybean varieties by crossing can increase drought resistance of other haplotype soybean varieties.

Example 2 functional verification of GmACO9 protein

The GmACO9 gene encodes an ACC oxidase which converts 1-aminocyclopropane carboxylic acid to ethylene by oxidation. The haplotype analysis result shows that the GmACO9 has 3 non-synonymous mutation SNP in an exon, and four haplotypes are formed (figure 1 e), wherein the drought-resistant phenotype of the material represented by Hap3 is optimal, and the material is a dominant haplotype. To further verify the role of GmACO9 in soybean drought stress resistance, williams 82, commonly used in the laboratory, was selected, and when the first round of three-leaf multi-leaf fully developed, 15% PEG solution was used to simulate drought, treat soybean material, and sample roots. The results showed that the expression level of GmACO9 gradually increased during the treatment, indicating that GmACO9 was induced by osmotic stress and was likely to function early in drought stress signal transduction (fig. 1 f).

In order to determine whether changes in SNP affect the enzymatic activity of the protein, the inventors detected two haplotypes, i.e., gmACO9, using an in vitro method ^Hap3 (dominant haplotype) and GmACO9 ^Hap1 (haplotype of GmACO9 in transgene acceptor DN 50). First, the protein is induced and purified in E.coli BL21 by in vitro induction of the protein, in tandem with the MBP tag. ACO enzyme activity was identified in vitro according to the method in the literature of Bulens et al (Bulens et al 2011) and ethylene release was determined using gas chromatography. The results show that both haplotypes Hap1 and Hap3 of GmACO9 have ACC oxidase activity, and that GmACO9 has the same substrate concentration ^Hap3 Catalytic activity higher than GmACO9 ^Hap1 The ethylene release amount is higher than GmACO9 ^Hap1 (FIG. 1 g). The specific operation steps are as follows:

pMal-c2x-GmACO9 vector construction, namely prokaryotic expression GmACO9 protein vector construction

1. According to the existing genotype information of the laboratory, selecting corresponding materials to amplify different GmACO9 haplotypes, separating leaves of different varieties of soybeans from plants, extracting RNA by using a total RNA extraction kit of the Viola plant, and carrying out reverse transcription by using a full-gold one-step cDNA synthesis kit to obtain cDNA of the corresponding materials.

2. Amplification of two haplotypes GmACO9 using primer pairs SEQ ID NO 13 and SEQ ID NO 14 ^Hap1 And GmACO9 ^Hap3 The sequences of the obtained PCR amplified products are shown as SEQ ID NO.5 and SEQ ID NO. 7 respectively

3. Construction of pMal-c2x-GmACO9 by homologous recombination ^Hap1 And pMal-c2x-GmACO9 ^Hap3 (FIGS. 2a, b), the specific procedure is shown inSeamless Cloning and Assembly Kit (full gold, cat: CU 101-01). The pMal-c2x vector is purchased from China plasmid vector strain cell gene collection center, and the vector carries a maltose binding protein tag and can be used for protein purification.

4. Introducing the constructed vector into Escherichia coli BL21 strain purchased from Tiangen Biochemical technology (Beijing) limited company by heat shock method, selecting positive colony, and performing first generation sequencing to obtain pMal-c2x-GmACO9 containing correct sequence ^Hap1 And pMal-c2x-GmACO9 ^Hap3 BL21 colonies of the vector.

Protein-induced purification

1. Inoculating the strain containing the target vector into a liquid LB culture medium containing Amp (ampicillin) resistance according to a ratio of 1:1000, and shaking overnight at 37 ℃;

2. an appropriate amount of overnight shaken bacteria was inoculated into 200mL of liquid LB containing the corresponding resistance, and the initial OD was adjusted ₆₀₀ 0.1-0.2. Culturing at 37deg.C for about 3 hr to OD 0.6;

3. adding IPTG to make the final concentration of the protein be 0.4mM, and carrying out protein induction for 18h under the condition of 160rpm at 16 ℃;

centrifuging at 5000rpm for 5min at 4.4 ℃ to collect thalli;

5. preparing purification, namely preparing column passing buffer solution: 20mM Tris-HCl (pH=7.4), 0.2M NaCl, 1mM EDTA,1mM DTT;

6. adding 25mL of the collected thalli into a column buffer solution, and performing ultrasonic crushing by using an ultrasonic instrument until the thalli becomes transparent from turbidity;

7. centrifuging the liquid after ultrasonic treatment at 10000rpm for 30min at 4 ℃;

8. taking 2mL of starch resin for column packing, flowing the protecting solution, and washing the balance column material with 10mL of column passing buffer solution for later use;

9. filtering the supernatant after centrifugation at 4 ℃ and slowly flowing through 1mL of starch resin;

10. washing the column with 10 times of column volume of column passing buffer solution, and collecting the impurity proteins which are not combined with the column material for subsequent electrophoresis analysis;

11. adding 10mM maltose into the column buffer solution, uniformly mixing, eluting target protein, eluting for several times, adding 500 mu L of eluent each time, and dividing into 6 tubes;

12. detection was performed using SDS-PAGE protein gel.

In vitro enzyme activity assay of GmACO9

1. Preparing MOPS reaction buffer solution: 1.046g MOPS,0.099g ascorbate, 0.168g sodium bicarbonate, 0.3mg ferrous sulfate, 0.01g ACC and 0.015g DTT are weighed, 50mL deionized water is added, the mixture is fully dissolved, 10mL glycerol is added, the pH is adjusted to 7.2, and the volume is fixed to 100mL.

2. 7.2mL of MOPS reaction buffer was added, 10. Mu.L of purified protein was added, and the lid was quickly closed. Vortex mixing the reaction solution and enzyme solution;

shaking table at 3.30 ℃ and shaking and incubating for 1h at 100 rpm;

4. vortex to release the synthesized ethylene in solution into the tube;

5. the ethylene content was measured by gas chromatography by sucking 1mL of the upper layer gas.

Example 3 transgenic phenotype statistics of GmACO9

Construction of recombinant plasmid

1. According to the genotype information existing in the laboratory, selecting a corresponding material to amplify GmACO9 ^Hap3 Haplotypes. Separating leaves of corresponding soybeans from plants, extracting RNA, and carrying out reverse transcription by using a full-gold one-step cDNA synthesis kit to obtain cDNA of corresponding materials.

2. Amplification of haplotype GmACO9 Using primer pairs SEQ ID NO. 15 and SEQ ID NO. 16 ^Hap3 The obtained PCR amplified product sequence SEQ ID NO. 7 shows

3. Construction of pTF101-35s-GmACO9 by homologous recombination ^Hap3 The specific operation steps are as followsSeamless Cloning and Assembly Kit (full gold, cat: CU 101-01). PTF101.1 vector was purchased from China center for type plasmid vector strain cell gene collection, and a map thereof is shown in FIG. 3a.

4. Recombinant plasmid pTF101-35s-GmACO9 ^Hap3 The recombinant agrobacterium is obtained by introducing agrobacterium strain EHA101 (purchased from the chinese plasmid vector strain cell gene collection center) and introducing the recombinant plasmid into agrobacterium strain EHA 101. The specific operation steps are as follows:

(1) About 150ng of plasmid was added to the competence of Agrobacterium strain EHA101 and left in ice for 30 minutes;

(2) The electrode cup is placed in an ultra-clean bench to be blown until no alcohol exists, and then is placed in ice to be cooled for standby;

(3) Adding the EHA101 competence mixed with the plasmid into an electrode cup, and shocking the electrode cup by a shocking instrument;

(4) 700 mu L of LB culture medium without antibiotics is added into EHA101 competence after electric shock, the mixture is placed on a shaking table at 30 ℃ and activated for 1h at 220rpm, then the mixture is coated on a Kan (kanamycin), spe (spectinomycin) and Rif (rifampicin) resistant culture medium, the culture is carried out for 2 to 3 days, the colony PCR identifies correct bacterial plaque, and the recombinant agrobacterium to be transferred is obtained after reactivation.

5. Amplifying target fragments by using the cDNA obtained in the step 1 as a template and using primer sequences in SEQ ID NO. 17 and SEQ ID NO. 18, constructing a GmACO9 RNAi vector, namely pFGC5941-GmACO9, by using a digestion connection method, wherein the map is shown in figure 3b, and introducing the recombinant plasmid into an agrobacterium strain EHA105 to obtain recombinant agrobacterium, and the specific operation is shown in the step 4.

2. Cotyledonary node transformation method for transforming soybean

The recombinant agrobacterium obtained in step 4 and step 5 was transformed into a recipient plant DN50 by a cotyledonary node transformation method (see Zhang et al, 2022 for specific methods of operation), and T was harvested ₀ Seed generation. Detailed operationThe method comprises the following steps:

1. seed sterilization and germination

DN50 soybean seeds with full round seeds and smooth surface and no disease spots are selected in a 120mm culture dish. Placing the culture dish into a dryer, placing a 250mL beaker into the dryer, adding 100mL10% sodium hypochlorite solution, slowly adding 4mL concentrated hydrochloric acid along the beaker, immediately covering the cover of the dryer, sterilizing soybean seeds by using chlorine for 18h, and uncovering the cover in an ultra clean bench after sterilization to blow off residual chlorine. The sterilized soybean seeds were placed evenly in germination medium (purchased from Coolaber company, PM 1062) with the umbilicus down, 30-35 seeds per dish. Then wrapping with fresh-keeping bag, cutting off ventilation opening, placing into dark incubator, germinating at 22deg.C for more than 16 hr.

2. Infection of Agrobacterium and co-cultivation of explants

Taking germinated seeds, firstly cutting off a part of cotyledons, longitudinally cutting the seeds into two symmetrical parts along the hypocotyl, gently scraping off a pair of true leaves at cotyledonary nodes under a microscope, and finally gently pricking the cotyledon nodes with a surgical knife to obtain the explant for transformation. Respectively taking EHA101 and EHA105 recombinant agrobacteria obtained in the steps 5 and 6 of construction of the recombinant plasmid stored in glycerol frozen at-80 ℃ and thawing on ice, dipping a small amount of bacterial liquid in an ultra clean bench, drawing lines, culturing on a YEP solid culture medium containing Kan (kanamycin) and Rif (rifampicin), activating and culturing for 2 days at 28 ℃, then coating on a new YEP solid culture medium containing Kan and Rif by using a coater, culturing overnight, and finally re-suspending the agrobacterium cultured overnight to OD by using a liquid co-culture medium ₆₀₀ The value is 0.6. Placing the prepared explant for transformation into resuspended agrobacterium tumefaciens bacteria solution, placing the explant into a dark incubator at 22 ℃ for infection overnight, then sucking the superfluous bacteria solution on the surface by using sterile filter paper, spreading cotyledonary node on a solid co-culture medium paved with the sterile filter paper, and carrying out dark infection at 22 ℃ for 5 days.

Wherein, the components of the liquid co-culture medium are as follows:

3. transgenic seedling acquisition

The cotyledonary node after co-culture for 5 days is obliquely inserted into a bud induction culture medium I (SI-I), the cotyledonary node is dark under the condition of 25 ℃ and 16h illumination for 8h, the illumination intensity is 5000-6000Lux, the culture is resumed for 7 days, the cotyledonary node is cut off too long and then is transferred into a bud induction culture medium II (SI-II) containing 8mg/mL of PPT (glufosinate), and the culture is continued for 14-20 days. The cluster buds are excised from the hypocotyl and transferred into bud elongation medium (SEM) containing 4mg/mL PPT, at 25 ℃ for 8 hours under illumination in the dark, with the illumination intensity of 5000-6000Lux, and subcultured every 10 days until the buds are elongated to about 5 cm. Cutting off the buds extending to about 5cm, inserting into rooting culture medium, irradiating for 8h at 25deg.C for darkness, and irradiating with 5000-6000Lux until the roots extend to 3-4cm, and preparing for transplanting.

In this step, the composition of the shoot induction medium I was B-5 salt (Beijing Mejie technologies Co., ltd.), B5 vitamin (Beijing Mejie technologies Co., ltd.), 30g/L sucrose, 0.6g/L MES (Sigma), 1.6 mg/L6-BA (6-benzylaminopurine, biological engineering (Shanghai) Co., ltd., A600743-0025), 50mg/L Cef (cefotaxime sodium, shanghai Altin Biotechnology Co., ltd.), 150mg/L Tim (Temerin/Temeiding, beijing Mejie technologies Co., ltd.), 4g/L glufosinate, 0.2% (w/v) vegetable gel (Sigma), pH 5.7; the composition of the bud induction culture medium II comprises B-5 salt, B5 vitamin, 30g/L sucrose, 0.6g/L MES,1.6 mg/L6-BA, 50mg/L Cef,150mg/L Tim,8g/L glufosinate, 0.2% (w/v) plant gel and pH 5.7; the composition of the bud elongation culture medium comprises MS salt, B5 vitamin, 30g/L sucrose, 0.6g/L MES,0.5mg/L gibberellin GA3 (manufactured by biological engineering (Shanghai) Co., ltd.), 1mg/L ZR (Beijing Simmondsia, tech Co., ltd.), 50mg/L L-Glu (Sigma), 50mg/L Asp (Sigma), 0.1mg/L IAA (Sigma), 50mg/L Cef,100mg/L Tim,4g/L glufosinate, 0.2% (w/v) plant gel, and pH 5.8; the rooting medium consists of MS salt, B5 vitamin, 20g/L sucrose, 0.6g/L MES,50mg/L L-Glu,50mg/L Asp,1.5mg/L IBA (Sigma), 25mg/L Tim,0.2% (w/v) plant gel and pH 5.8.

4. Seedling hardening, transplanting and screening

Removing a sealing film from tissue culture seedlings to be transplanted, adding a small amount of sterile water, darkening at 25 ℃ for 16h under the illumination of 8h, and culturing for two days, transplanting the seedlings, uniformly mixing vermiculite and turfy soil in equal quantity, placing the mixture into a tray with water, extracting the tissue culture seedlings from a rooting culture medium, flushing the residual culture medium at the root, and transferring the culture medium into nutrient soil fully absorbing water. Soybean leaves were coated with 0.1% Basta herbicide and after 3 days no yellowing response was seen as a transgenic positive plant.

Subsequent T ₁ The generation and the subsequent passage of the transgenic strain are sprayed with 0.1% Basta herbicide for screening to obtain 2 recombinant plasmids pTF101-35s-GmACO9 successfully transferred ^Hap3 Transgenic plants (i.e.plants OE1 overexpressing GmACO 9) ^GmACO9 And OE2 ^GmACO9 ) The expression quantity of GmACO9 genes in transgenic plants is obviously improved; at the same time, the transgenic plant (namely RNAi transgenic plant, RNAi-1) successfully transferred into the recombinant plasmid pFGC5941-GmACO9 is obtained ^GmACO9 And RNAi-2 ^GmACO9 ) The expression level of GmACO9 was significantly suppressed in RNAi transgenic plants (fig. 4 c).

3. Drought-resistant phenotype identification of GmACO9 transgenic strain

This example demonstrates the successful transfer of the recombinant plasmid pTF101-35s-GmACO9 into DN50 ^Hap3 The drought resistance of the obtained over-expression transgenic plant is obviously improved. The RNAi transgenic plants obtained after successful transfer of the recombinant plasmid pFGC5941-GmACO9 in DN50 showed drought sensitivity (FIG. 4).

In particular, the transgenic lines are obtained according to the steps shown above, and T is identified by using a water-break treatment method ₂ The phenotype of the transgenic plant comprises the following specific operation steps:

1. filtering the nutrient soil by using a mesh screen with the size of 1.5mm, and uniformly mixing the nutrient soil with vermiculite according to the volume ratio of 1:2;

2. material planting is carried out by using small square boxes with the size of 8cm multiplied by 8cm, the boxes are filled with soil (120 g) with the same mass, each 18 small square boxes are placed in a seedling raising tray with the size of 5.4cm multiplied by 2.8cm multiplied by 6.5cm, 9 small square boxes are used for planting transgenic plants, 9 transgenic plants are planted with DN50, and 4 repeated seedlings are planted for each transgenic plant line, namely 4 seedling raising trays;

3. sowing, and carrying out positive plant screening by spraying glufosinate-ammonium (Basta) on the transgenic material when true leaves are unfolded;

4. after the first round of three-leaf compound leaves are fully unfolded, selecting positive transgenic plants, watering until saturation, and cutting off water and starting drought treatment;

5. photographing 12 days of drought treatment, rehydrating 14 days of drought treatment, and counting the survival rate of transgenic lines and DN50 in each breeding tray after rehydrating for 3 days.

Wherein, the phenotype of wild type DN50 and GmACO9 transgenic plants under normal growth conditions has no obvious difference (figure 4 a), but compared with wild type DN50 after 12 days of water-cut treatment, RNAi transgenic plants show wilting caused by water shortage, and after 14 days of water-cut treatment, overexpression plants show obvious drought resistance (figure 4 b). Rehydration was performed after 14 days of water-break treatment, and survival rates of wild-type DN50 and transgenic lines were counted after 3 days of rehydration, which indicated that the survival rate of the overexpressing transgenic lines was significantly higher than that of the wild-type DN50, whereas the survival rate of RNAi transgenic plants was significantly lower than that of the wild-type DN50 (FIGS. 4d, e).

The results show that the GmACO9 gene is a key gene for regulating the drought resistance of soybeans, and the drought resistance of soybeans can be improved by over-expressing the GmACO9 gene in soybean plants.

The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.

Reference to the literature

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Sequence listing

SEQ ID NO:1GmACO9-Hap1 307aa protein Glycine max (Soybean)

MEIPVIDFSNLNGDKRGDTMALLHEACEKWGCFMVENHEIDTQLMEKLKQLINTYYEEDLKESFYQSEIAKRLEKQQNTSDIDWEITFFIWHRPTSNINEIPNISRELCQTMDEYIAQLLKLGEKLSELMSENLGLEKDYIKKAFSGSGEGPAVGTKVAKYPQCPRPELVRGLREHTDAGGIILLLQDDKVPGLEFFKDGKWVEIPPPKNNAVFVNTGDQVEVLSNGLYKSVLHRVMPDNSGSRTSIATFYNPIGDAIISPAPKLLYPSNFRYGDYLKLYGSTKFGEKAPRFECMKNMTNGHKNIPA

SEQ ID NO. 2GmACO9-Hap2 307aa protein Glycine max (Soybean)

MEIPVIDFSNLNGDKRGDTMALLHEACEKWGCFMVENHEIDTQLMEKLKQLINTYYEEDLKESFYQSEIAKRLEKEQNTSDIDWEITFFIWHRPTSNINEIPNISRELCQTMDEYIAQLLKLGEKLSELMSENLGLEKDYIKKAFSGSGEGPAVGTKVAKYPQCPRPELVRGLREHTDAGGIILLLQDDKVPGLELFKDGKWVEIPPPKNNAVFVNTGDQVEVLSNGLYKSVVHRVMPDNSGSRTSIATFYNPIGDAIISPAPKLLYPSNFRYGDYLKLYGSTKFGEKAPRFECMKNMTNGHKNIPA

SEQ ID NO:3GmACO9-Hap3 307aa protein Glycine max (Soybean)

MEIPVIDFSNLNGDKRGDTMALLHEACEKWGCFMVENHEIDTQLMEKLKQLINTYYEEDLKESFYQSEIAKRLEKEQNTSDIDWEITFFIWHRP TSNINEIPNISRELCQTMDEYIAQLLKLGEKLSELMSENLGLEKDYIKKAFSGSGEGPAVGTKVAKYPQCPRPELVRGLREHTDAGGIILLLQDDKVPGLEFFKDGKWVEIPPPKNNAVFVNTGDQVEVLSNGLYKSVLHRVMPDNSGSRTSIATFYNPIGDAIISPAPKLLYPSNFRYGDYLKLYGSTKFGEKAPRFECMKNMTNGHKNIPA

SEQ ID NO. 4GmACO9-Hap4 307aa protein Glycine max (Soybean)

MEIPVIDFSNLNGDKRGDTMALLHEACEKWGCFMVENHEIDTQLMEKLKQLINTYYEEDLKESFYQSEIAKRLEKEQNTSDIDWEITFFIWHRPTSNINEIPNISRELCQTMDEYIAQLLKLGEKLSELMSENLGLEKDYIKKAFSGSGEGPAVGTKVAKYPQCPRPELVRGLREHTDAGGIILLLQDDKVPGLEFFKDGKWVEIPPPKNNAVFVNTGDQVEVLSNGLYKSVVHRVMPDNSGSRTSIATFYNPIGDAIISPAPKLLYPSNFRYGDYLKLYGSTKFGEKAPRFECMKNMTNGHKNIPA

SEQ ID NO. 5GmPrx16-Hap1 924bp cDNA Glycine max (Soybean)

ATGGAGATCCCTGTGATAGATTTTAGTAACCTCAATGGGGACAAGAGGGGTGACACAATGGCCCTATTGCACGAAGCTTGTGAAAAATGGGGCTGCTTTATGGTTGAGAACCATGAAATTGACACACAACTGATGGAGAAGTTGAAGCAGTTAATCAATACATACTATGAGGAAGACCTAAAGGAAAGCTTCTACCAGTCTGAGATAGCCAAGAGGTTGGAGAAACAGCAGAACACCTCTGATATAGATTGGGAAATTACCTTCTTCATTTGGCATCGCCCCACCTCTAACATCAATGAAATTCCAAACATCTCTCGGGAGCTTTGCCAAACAATGGATGAGTACATTGCACAGCTCCTGAAGCTGGGAGAGAAGTTATCTGAGCTCATGAGTGAAAATCTTGGTTTGGAGAAGGATTACATCAAGAAAGCATTTTCTGGAAGTGGTGAGGGTCCTGCTGTGGGAACAAAAGTGGCCAAGTACCCTCAGTGTCCACGTCCAGAACTTGTGAGGGGACTTAGAGAGCACACAGATGCGGGTGGCATCATTCTACTGCTCCAAGATGACAAAGTGCCTGGTCTGGAATTCTTCAAAGATGGCAAATGGGTCGAGATTCCACCACCCAAGAACAATGCCGTTTTTGTGAACAC GGGTGATCAAGTGGAAGTGTTGAGCAATGGCTTATATAAAAGTGTTCTGCATAGGGTCATGCCTGACAATAGTGGAAGCAGAACCTCCATTGCTACCTTTTATAATCCCATTGGAGATGCCATTATTTCCCCAGCTCCTAAGCTCTTGTACCCAAGCAATTTCCGTTATGGGGACTATTTGAAGCTCTATGGCAGCACCAAGTTCGGTGAAAAGGCTCCTCGATTTGAATGCATGAAGAACATGACCAATGGCCATAAAAATATTCCAGCTTGA

SEQ ID NO. 6GmACO9-Hap2 924bp cDNA Glycine max (Soybean)

ATGGAGATCCCTGTGATAGATTTTAGTAACCTCAATGGGGACAAGAGGGGTGACACAATGGCCCTATTGCACGAAGCTTGTGAAAAATGGGGCTGCTTTATGGTTGAGAACCATGAAATTGACACACAACTGATGGAGAAGTTGAAGCAGTTAATCAATACATACTATGAGGAAGACCTAAAGGAAAGCTTCTACCAGTCTGAGATAGCCAAGAGGTTGGAGAAAGAGCAGAACACCTCTGATATAGATTGGGAAATTACCTTCTTCATTTGGCATCGCCCCACCTCTAACATCAATGAAATTCCAAACATCTCTCGGGAGCTTTGCCAAACAATGGATGAGTACATTGCACAGCTCCTGAAGCTGGGAGAGAAGTTATCTGAGCTCATGAGTGAAAATCTTGGTTTGGAGAAGGATTACATCAAGAAAGCATTTTCTGGAAGTGGTGAGGGTCCTGCTGTGGGAACAAAAGTGGCCAAGTACCCTCAGTGTCCACGTCCAGAACTTGTGAGGGGACTTAGAGAGCACACAGATGCGGGTGGCATCATTCTACTGCTCCAAGATGACAAAGTGCCTGGTCTGGAACTCTTCAAAGATGGCAAATGGGTCGAGATTCCACCACCCAAGAACAATGCCGTTTTTGTGAACACGGGTGATCAAGTGGAAGTGTTGAGCAATGGCTTATATAAAAGTGTTGTGCATAGGGTCATGCCTGACAATAGTGGAAGCAGAACCTCCATTGCTACCTTTTATAATCCCATTGGAGATGCCATTATTTCCCCAGCTCCTAAGCTCTTGTACCCAAGCAATTTCCGTTATGGGGACTATTTGAAGCTCTATGGCAGCACCAAGTTCGGTGAAAAGGCTCCTCGATTTGAATGCATGAAGAACATGACCAATGGCCATAAAAATATTCCAGCTTGA

SEQ ID NO. 7GmACO9-Hap3 924bp cDNA Glycine max (Soybean)

ATGGAGATCCCTGTGATAGATTTTAGTAACCTCAATGGGGACAAGAGGGGTGACACAATGGCCCTATTGCACGAAGCTTGTGAAAAATGGGGCTGCTTTATGGTTGAGAACCATGAAATTGACACACAACTGATGGAGAAGTTGAAGCAGTTAATCAATACATACTATGAGGAAGACCTAAAGGAAAGCTTCTACCAGTCTGAGATAGCCAAGAGGTTGGAGAAAGAGCAGAACACCTCTGATATAGATTGGGAAATTACCTTCTTCATTTGGCATCGCCCCACCTCTAACATCAATGAAATTCCAAACATCTCTCGGGAGCTTTGCCAAACAATGGATGAGTACATTGCACAGCTCCTGAAGCTGGGAGAGAAGTTATCTGAGCTCATGAGTGAAAATCTTGGTTTGGAGAAGGATTACATCAAGAAAGCATTTTCTGGAAGTGGTGAGGGTCCTGCTGTGGGAACAAAAGTGGCCAAGTACCCTCAGTGTCCACGTCCAGAACTTGTGAGGGGACTTAGAGAGCACACAGATGCGGGTGGCATCATTCTACTGCTCCAAGATGACAAAGTGCCTGGTCTGGAATTCTTCAAAGATGGCAAATGGGTCGAGATTCCACCACCCAAGAACAATGCCGTTTTTGTGAACACGGGTGATCAAGTGGAAGTGTTGAGCAATGGCTTATATAAAAGTGTTCTGCATAGGGTCATGCCTGACAATAGTGGAAGCAGAACCTCCATTGCTACCTTTTATAATCCCATTGGAGATGCCATTATTTCCCCAGCTCCTAAGCTCTTGTACCCAAGCAATTTCCGTTATGGGGACTATTTGAAGCTCTATGGCAGCACCAAGTTCGGTGAAAAGGCTCCTCGATTTGAATGCATGAAGAACATGACCAATGGCCATAAAAATATTCCAGCTTGA

SEQ ID NO. 8GmACO9-Hap4 924bp cDNA Glycine max (Soybean)

ATGGAGATCCCTGTGATAGATTTTAGTAACCTCAATGGGGACAAGAGGGGTGACACAATGGCCCTATTGCACGAAGCTTGTGAAAAATGGGGCTGCTTTATGGTTGAGAACCATGAAATTGACACACAACTGATGGAGAAGTTGAAGCAGTTAATCAATACATACTATGAGGAAGACCTAAAGGAAAGCTTCTACCAGTCTGAGATAGCCAAGAGGTTGGAGAAAGAGCAGAACACCTCTGATATAGATTGGGAAATTACCTTCTTCATTTGGCATCGCCCCACCTCTAACATCAATGAAATTCCAAACATCTCTCGGGAGCTTTGCCAAACAATGGATGAGTACATTGCACAGCTCCTGAAGCTGGGAGA GAAGTTATCTGAGCTCATGAGTGAAAATCTTGGTTTGGAGAAGGATTACATCAAGAAAGCATTTTCTGGAAGTGGTGAGGGTCCTGCTGTGGGAACAAAAGTGGCCAAGTACCCTCAGTGTCCACGTCCAGAACTTGTGAGGGGACTTAGAGAGCACACAGATGCGGGTGGCATCATTCTACTGCTCCAAGATGACAAAGTGCCTGGTCTGGAATTCTTCAAAGATGGCAAATGGGTCGAGATTCCACCACCCAAGAACAATGCCGTTTTTGTGAACACGGGTGATCAAGTGGAAGTGTTGAGCAATGGCTTATATAAAAGTGTTGTGCATAGGGTCATGCCTGACAATAGTGGAAGCAGAACCTCCATTGCTACCTTTTATAATCCCATTGGAGATGCCATTATTTCCCCAGCTCCTAAGCTCTTGTACCCAAGCAATTTCCGTTATGGGGACTATTTGAAGCTCTATGGCAGCACCAAGTTCGGTGAAAAGGCTCCTCGATTTGAATGCATGAAGAACATGACCAATGGCCATAAAAATATTCCAGCTTGA

SEQ ID NO 9GmACO9-Hap1 1606bp gDNA Glycine max (Soybean)

TTTCGCCCTATATATACCCCATCCTCTGTAACAAACCTTCTACAAAGAAACATTAAGTATCTAGTCTAGTATACAAAGATGGAGATCCCTGTGATAGATTTTAGTAACCTCAATGGGGACAAGAGGGGTGACACAATGGCCCTATTGCACGAAGCTTGTGAAAAATGGGGCTGCTTTATGGTATGGTATATAGAGAACACAAATTATATATGTTGTCCCTTTGTTTTTCTATTCTTCATCATTTCTCAATTAATTTTCTTCTTTAACACATTTGCATATGCAGGTTGAGAACCATGAAATTGACACACAACTGATGGAGAAGTTGAAGCAGTTAATCAATACATACTATGAGGAAGACCTAAAGGAAAGCTTCTACCAGTCTGAGATAGCCAAGAGGTTGGAGAAACAGCAGAACACCTCTGATATAGATTGGGAAATTACCTTCTTCATTTGGCATCGCCCCACCTCTAACATCAATGAAATTCCAAACATCTCTCGGGAGCTTTGGTAAGTCAATCCATATACGTGACTTTTTTTTTCCTACATGTATTGTCCTTCAGAGATGATTTTGACCGAGATACAGTTGAATATTAAATGTGAATATCTCTCCTAATATAAATTTAAACTTTGACTTACCATGTCGTGGAAACTAATTAACCCCTTCAGCAGTTCTGCTACCAACCCTTGTTGATTCATTTGTGGCATCTATAGAAAACTTATATTAGGAGTCCTGGCAACACTAAAAGAGAGTAGAATGGAAAG AGAATGTTTCAGTTATATTAAAAATGTTAAGAGTGTGTTGTAAACATATTTTGGCATGATATATAAGTGTATATGTAATTAAATGCAGCCAAACAATGGATGAGTACATTGCACAGCTCCTGAAGCTGGGAGAGAAGTTATCTGAGCTCATGAGTGAAAATCTTGGTTTGGAGAAGGATTACATCAAGAAAGCATTTTCTGGAAGTGGTGAGGGTCCTGCTGTGGGAACAAAAGTGGCCAAGTACCCTCAGTGTCCACGTCCAGAACTTGTGAGGGGACTTAGAGAGCACACAGATGCGGGTGGCATCATTCTACTGCTCCAAGATGACAAAGTGCCTGGTCTGGAATTCTTCAAAGATGGCAAATGGGTCGAGATTCCACCACCCAAGAACAATGCCGTTTTTGTGAACACGGGTGATCAAGTGGAAGTGTTGAGCAATGGCTTATATAAAAGTGTTCTGCATAGGGTCATGCCTGACAATAGTGGAAGCAGAACCTCCATTGCTACCTTTTATAATCCCATTGGAGATGCCATTATTTCCCCAGCTCCTAAGCTCTTGTACCCAAGCAATTTCCGTTATGGGGACTATTTGAAGCTCTATGGCAGCACCAAGTTCGGTGAAAAGGCTCCTCGATTTGAATGCATGAAGAACATGACCAATGGCCATAAAAATATTCCAGCTTGAGACTTAGAAGACTTGGAAGAACGTCGCATTATTACCCTTGCTTGGGATAGCTAAACTTACAAGTTATTATTATGTTTACATTTGGAATGGTAATATTTCTTTTTTGTTCTATGGCTTGATTCCACAAATAGAAAAATTAAGGGTTAAGATTTATCTAG

SEQ ID NO 10GmACO9-Hap2 1606bp gDNA Glycine max (Soybean)

TTTCGCCCTATATATACCCCATCCTCTGTAACAAACCTTCTACAAAGAAACATTAAGTATCTAGTCTAGTATACAAAGATGGAGATCCCTGTGATAGATTTTAGTAACCTCAATGGGGACAAGAGGGGTGACACAATGGCCCTATTGCACGAAGCTTGTGAAAAATGGGGCTGCTTTATGGTATGGTATATAGAGAACACAAATTATATATGTTGTCCCTTTGTTTTTCTATTCTTCATCATTTCTCAATTAATTTTCTTCTTTAACACATTTGCATATGCAGGTTGAGAACCATGAAATTGACACACAACTGATGGAGAAGTTGAAGCAGTTAATCAATACATACTATGAGGAAGACCTAAAGGAAAGCTTCTACCAGTCTGAGATAGCCAAGAGGTTGGAGAAAGAGCAGAACACCTCTGATATAGATTGGGAAATTACCTTCTTCATTTGGCATCGCCCCACCTCTAACA TCAATGAAATTCCAAACATCTCTCGGGAGCTTTGGTAAGTCAATCCATATACGTGACTTTTTTTTTCCTACATGTATTGTCCTTCAGAGATGATTTTGACCGAGATACAGTTGAATATTAAATGTGAATATCTCTCCTAATATAAATTTAAACTTTGACTTACCATGTCGTGGAAACTAATTAACCCCTTCAGCAGTTCTGCTACCAACCCTTGTTGATTCATTTGTGGCATCTATAGAAAACTTATATTAGGAGTCCTGGCAACACTAAAAGAGAGTAGAATGGAAAGAGAATGTTTCAGTTATATTAAAAATGTTAAGAGTGTGTTGTAAACATATTTTGGCATGATATATAAGTGTATATGTAATTAAATGCAGCCAAACAATGGATGAGTACATTGCACAGCTCCTGAAGCTGGGAGAGAAGTTATCTGAGCTCATGAGTGAAAATCTTGGTTTGGAGAAGGATTACATCAAGAAAGCATTTTCTGGAAGTGGTGAGGGTCCTGCTGTGGGAACAAAAGTGGCCAAGTACCCTCAGTGTCCACGTCCAGAACTTGTGAGGGGACTTAGAGAGCACACAGATGCGGGTGGCATCATTCTACTGCTCCAAGATGACAAAGTGCCTGGTCTGGAACTCTTCAAAGATGGCAAATGGGTCGAGATTCCACCACCCAAGAACAATGCCGTTTTTGTGAACACGGGTGATCAAGTGGAAGTGTTGAGCAATGGCTTATATAAAAGTGTTGTGCATAGGGTCATGCCTGACAATAGTGGAAGCAGAACCTCCATTGCTACCTTTTATAATCCCATTGGAGATGCCATTATTTCCCCAGCTCCTAAGCTCTTGTACCCAAGCAATTTCCGTTATGGGGACTATTTGAAGCTCTATGGCAGCACCAAGTTCGGTGAAAAGGCTCCTCGATTTGAATGCATGAAGAACATGACCAATGGCCATAAAAATATTCCAGCTTGAGACTTAGAAGACTTGGAAGAACGTCGCATTATTACCCTTGCTTGGGATAGCTAAACTTACAAGTTATTATTATGTTTACATTTGGAATGGTAATATTTCTTTTTTGTTCTATGGCTTGATTCCACAAATAGAAAAATTAAGGGTTAAGATTTATCTAG

SEQ ID NO. 11GmACO9-Hap3 1606bp gDNA Glycine max (Soybean)

TTTCGCCCTATATATACCCCATCCTCTGTAACAAACCTTCTACAAAGAAACATTAAGTATCTAGTCTAGTATACAAAGATGGAGATCCCTGTGATAGATTTTAGTAACCTCAATGGGGACAAGAGGGGTGACACAATGGCCCTATTGCACGAAGCTTGTGAAAAATGGGGCTGCTTTATGGTATGGTA

TATAGAGAACACAAATTATATATGTTGTCCCTTTGTTTTTCTATTCTTC

ATCATTTCTCAATTAATTTTCTTCTTTAACACATTTGCATATGCAGGTT

GAGAACCATGAAATTGACACACAACTGATGGAGAAGTTGAAGCAGT

TAATCAATACATACTATGAGGAAGACCTAAAGGAAAGCTTCTACCAG

TCTGAGATAGCCAAGAGGTTGGAGAAAGAGCAGAACACCTCTGATA

TAGATTGGGAAATTACCTTCTTCATTTGGCATCGCCCCACCTCTAACA

TCAATGAAATTCCAAACATCTCTCGGGAGCTTTGGTAAGTCAATCCAT

ATACGTGACTTTTTTTTTCCTACATGTATTGTCCTTCAGAGATGATTTT

GACCGAGATACAGTTGAATATTAAATGTGAATATCTCTCCTAATATAAA

TTTAAACTTTGACTTACCATGTCGTGGAAACTAATTAACCCCTTCAGC

AGTTCTGCTACCAACCCTTGTTGATTCATTTGTGGCATCTATAGAAAA

CTTATATTAGGAGTCCTGGCAACACTAAAAGAGAGTAGAATGGAAAG

AGAATGTTTCAGTTATATTAAAAATGTTAAGAGTGTGTTGTAAACATA

TTTTGGCATGATATATAAGTGTATATGTAATTAAATGCAGCCAAACAAT

GGATGAGTACATTGCACAGCTCCTGAAGCTGGGAGAGAAGTTATCTG

AGCTCATGAGTGAAAATCTTGGTTTGGAGAAGGATTACATCAAGAAA

GCATTTTCTGGAAGTGGTGAGGGTCCTGCTGTGGGAACAAAAGTGG

CCAAGTACCCTCAGTGTCCACGTCCAGAACTTGTGAGGGGACTTAG

AGAGCACACAGATGCGGGTGGCATCATTCTACTGCTCCAAGATGACA

AAGTGCCTGGTCTGGAATTCTTCAAAGATGGCAAATGGGTCGAGATT

CCACCACCCAAGAACAATGCCGTTTTTGTGAACACGGGTGATCAAG

TGGAAGTGTTGAGCAATGGCTTATATAAAAGTGTTCTGCATAGGGTCA

TGCCTGACAATAGTGGAAGCAGAACCTCCATTGCTACCTTTTATAATC

CCATTGGAGATGCCATTATTTCCCCAGCTCCTAAGCTCTTGTACCCAA

GCAATTTCCGTTATGGGGACTATTTGAAGCTCTATGGCAGCACCAAG

TTCGGTGAAAAGGCTCCTCGATTTGAATGCATGAAGAACATGACCAA

TGGCCATAAAAATATTCCAGCTTGAGACTTAGAAGACTTGGAAGAAC

GTCGCATTATTACCCTTGCTTGGGATAGCTAAACTTACAAGTTATTATT

ATGTTTACATTTGGAATGGTAATATTTCTTTTTTGTTCTATGGCTTGATT

CCACAAATAGAAAAATTAAGGGTTAAGATTTATCTAG

SEQ ID NO. 12GmACO9-Hap4 1606bp gDNA Glycine max (Soybean)

TTTCGCCCTATATATACCCCATCCTCTGTAACAAACCTTCTACAAAGAAACATTAAGTATCTAGTCTAGTATACAAAGATGGAGATCCCTGTGATAGATTTTAGTAACCTCAATGGGGACAAGAGGGGTGACACAATGGCCCTATTGCACGAAGCTTGTGAAAAATGGGGCTGCTTTATGGTATGGTATATAGAGAACACAAATTATATATGTTGTCCCTTTGTTTTTCTATTCTTCATCATTTCTCAATTAATTTTCTTCTTTAACACATTTGCATATGCAGGTTGAGAACCATGAAATTGACACACAACTGATGGAGAAGTTGAAGCAGTTAATCAATACATACTATGAGGAAGACCTAAAGGAAAGCTTCTACCAGTCTGAGATAGCCAAGAGGTTGGAGAAAGAGCAGAACACCTCTGATATAGATTGGGAAATTACCTTCTTCATTTGGCATCGCCCCACCTCTAACATCAATGAAATTCCAAACATCTCTCGGGAGCTTTGGTAAGTCAATCCATATACGTGACTTTTTTTTTCCTACATGTATTGTCCTTCAGAGATGATTTTGACCGAGATACAGTTGAATATTAAATGTGAATATCTCTCCTAATATAAATTTAAACTTTGACTTACCATGTCGTGGAAACTAATTAACCCCTTCAGCAGTTCTGCTACCAACCCTTGTTGATTCATTTGTGGCATCTATAGAAAACTTATATTAGGAGTCCTGGCAACACTAAAAGAGAGTAGAATGGAAAGAGAATGTTTCAGTTATATTAAAAATGTTAAGAGTGTGTTGTAAACATATTTTGGCATGATATATAAGTGTATATGTAATTAAATGCAGCCAAACAATGGATGAGTACATTGCACAGCTCCTGAAGCTGGGAGAGAAGTTATCTGAGCTCATGAGTGAAAATCTTGGTTTGGAGAAGGATTACATCAAGAAAGCATTTTCTGGAAGTGGTGAGGGTCCTGCTGTGGGAACAAAAGTGGCCAAGTACCCTCAGTGTCCACGTCCAGAACTTGTGAGGGGACTTAGAGAGCACACAGATGCGGGTGGCATCATTCTACTGCTCCAAGATGACAAAGTGCCTGGTCTGGAATTCTTCAAAGATGGCAAATGGGTCGAGATTCCACCACCCAAGAACAATGCCGTTTTTGTGAACACGGGTGATCAAGTGGAAGTGTTGAGCAATGGCTTATATAAAAGTGTTGTGCATAGGGTCATGCCTGACAATAGTGGAAGCAGAACCTCCATTGCTACCTTTTATAATCCCATTGGAGATGCCATTATTTCCCCAGCTCCTAAGCTCTTGTACCCAAGCAATTTCCGTTATGGGGACTATTTGAAGCTCTATGGCAGCACCAAGTTCGGTGAAAAGGCTCCTCGATTTGAATGCATGAAGAACATGACCAATGGCCATAAAAATATTCCAGCTTGAGACTTAGAAGACTTGGAAGAACGTCGCATTATTACCCTTGCTTGGGATAGCTAAACTTACAAGTTATTATTATGTTTACATTTGGAATGGTAATATTTCTTTTTTGTTCTATGGCTTGATTCCACAAATAGAAAAATTAAGGGTTAAGATTTATCTAG

SEQ ID NO. 13GmACO 9-MBP-F48 bp DNA Artificial (Artificial) synthetic primer

GGGAAGGATTTCAGAATTCGGAATGGAGATCCCTGTGATAGATTTTAG

14GmACO 9-MBP-R43 bp DNA Artificial (Artificial) synthetic primer

TAAAACGACGGCCAGTGCCATCAAGCTGGAATATTTTTATGGC

SEQ ID NO. 15GmACO 9-OE-F52 bp DNA Artificial (Artificial) synthetic primer

TTCATTTGGAGAGAACACGGGGGACATGGAGATCCCTGTGATAGATTTTAGT

SEQ ID NO. 16GmACO 9-OE-R49 bp DNA Artificial (Artificial) synthetic primer

TTGAACGATCGGGGAAATTCGAGCTTCAAGCTGGAATATTTTTATGGCC

17GmACO 9-RNAi-F36 bp DNA Artificial (Artificial) synthetic primer

CCATGGTCTAGACAATGAAATTCCAAACATCTCTCG

SEQ ID NO. 18GmACO 9-RNAi-R35 bp DNA Artificial (Artificial) synthetic primer

GGCGCGCCGGATCCAAAACGGCATTGTTCTTGGGT

Claims (8)

1. The amino acid sequence of the drought-resistant protein is shown as SEQ ID NO.1, SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4.

2. A polynucleotide sequence encoding the drought resistance protein of claim 1.

3. The polynucleotide sequence of claim 2 which is GmACO9 ^Hap1 Gene, gmACO9 ^Hap2 Gene, gmACO9 ^Hap3 Gene or GmACO9 ^Hap4 A gene, wherein the GmACO9 ^Hap1 Gene, gmACO9 ^Hap2 Gene, gmACO9 ^Hap3 Gene or GmACO9 ^Hap4 The cDNA sequence of the gene is shown as SEQ ID NO.5, SEQ ID NO. 6, SEQ ID NO. 7 or SEQ ID NO.8 respectively.

4. The polynucleotide sequence of claim 3, wherein the GmACO9 ^Hap1 Gene, gmACO9 ^Hap2 Gene, gmACO9 ^Hap3 Gene or GmACO9 ^Hap4 The gDNA sequence of the gene is shown as SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12 respectively.

5. A vector, wherein the vector comprises a polynucleotide sequence according to any one of claims 2-4, wherein the vector is a plant expression vector, preferably a binary agrobacterium vector.

6. A host cell comprising the polynucleotide sequence of any one of claims 2-4 or the vector of claim 5, wherein the host cell is selected from the group consisting of an Escherichia coli cell, an agrobacterium Agrobacterium tumefaciens cell, or a plant cell.

7. Use of the drought-resistance protein of claim 1, the polynucleotide sequence of any one of claims 2-4 for growing plants, preferably leguminous plants, more preferably soybean, with increased drought resistance.

8. A method of growing a transgenic plant with increased drought resistance comprising introducing into a plant cell or tissue of interest a drought resistance protein according to claim 1, a polynucleotide sequence according to any one of claims 2-4, or a vector according to claim 5 or a host cell according to claim 6, resulting in a transgenic plant with increased drought resistance compared to the plant of interest, said plant being a leguminous plant, preferably soybean.