Application of GmSUR2310 gene in improving flood tolerance and yield of soybeans

Abstract

The invention discloses an application of a GmSUR2310 gene in improving flood tolerance and yield of soybeans, and belongs to the technical field of plant genetic engineering and molecular breeding. The gene GmSUR2310 is overexpressed in plants (arabidopsis thaliana and soybean), so that the tolerance of the gene GmSUR2310 to waterflooding stress is remarkably improved. Meanwhile, the branch number, the pod number, the grain number and the yield of a single soybean plant can be increased, and the method has important significance on increasing the yield of the soybean. The results jointly show that the GmSUR2310 gene plays a key role in synergic improvement of plant flooding resistance and yield, and is a flooding-resistant breeding gene with important application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant genetic engineering and molecular breeding, and particularly relates to application of a GmSUR2 310 gene in improving soybean flooding tolerance and yield. BACKGROUND

[0002] Flood stress is one of the main abiotic stresses affecting global agricultural production. As an important oil and feed crop, soybean is highly sensitive to flood stress, and can result in yield loss of 17%-56% during the vegetative and reproductive growth stages. At present, the molecular mechanisms of soybean response to flood stress are not well understood, and the resources of key genes for molecular breeding improvement are very limited.

[0003] At present, about 27 flood-related quantitative trait loci (QTLs) have been located in the soybean genome, which are widely distributed on almost all chromosomes except chromosomes 16, 17 and 20. It is worth noting that a QTL located on chromosome 3 is confirmed to be related to root architecture under flood stress, and the tolerance allele of the locus can enhance the flooding tolerance of plants by promoting root growth. Among the 23 genes identified in the QTL region, the role of Gmly.03g031000 (named GmSUR2 310 ) in regulating soybean flooding tolerance has not been elucidated. Therefore, it has become a technical problem to be solved urgently to deeply analyze the major genes in the QTL region, reveal their functions and action pathways in the flooding tolerance physiological process, and effectively apply them to soybean flooding tolerance molecular breeding. SUMMARY

[0004] The purpose of the present application is to provide application of a GmSUR2 310 gene in improving soybean flooding tolerance and yield, so as to solve the problems in the prior art. The GmSUR2 310 gene positively regulates soybean flooding tolerance and yield, which provides a scientific basis for simultaneously improving the flood tolerance and yield of soybean.

[0005] To achieve the above purpose, the present application provides the following solutions.

[0006] The present application provides application of a GmSUR2 310 gene in any one of the following aspects:

[0007] (1) application in regulating flooding tolerance of plants;

[0008] (2) application in regulating branch number of plants;

[0009] (3) application in regulating pod number per plant;

[0010] (4) application in regulating grain number per plant of the plant;

[0011] (5) application in regulating yield per plant of the plant;

[0012] (6) application in cultivating the plant with flood tolerance;

[0013] The nucleotide sequence of the GmSUR2 310 gene is shown as SEQ ID NO. 1.

[0014] The application further provides application of the protein expressed by the GmSUR2 310 gene in any one of the following aspects:

[0015] (1) application in regulating flood tolerance of the plant;

[0016] (2) application in regulating branch number of the plant;

[0017] (3) application in regulating pod number per plant of the plant;

[0018] (4) application in regulating grain number per plant of the plant;

[0019] (5) application in regulating yield per plant of the plant;

[0020] (6) application in cultivating the plant with flood tolerance;

[0021] The nucleotide sequence of the GmSUR2 310 gene is shown as SEQ ID NO. 1, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

[0022] The application further provides application of the recombinant vector containing the GmSUR2 310 gene in any one of the following aspects:

[0023] (1) application in regulating flood tolerance of the plant;

[0024] (2) application in regulating branch number of the plant;

[0025] (3) application in regulating pod number per plant of the plant;

[0026] (4) application in regulating grain number per plant of the plant;

[0027] (5) application in regulating yield per plant of the plant;

[0028] (6) application in cultivating the plant with flood tolerance;

[0029] The nucleotide sequence of the GmSUR2 310The gene GmSUR2 is introduced into an expression vector to construct a recombinant vector. 310 The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

[0030] The application also provides application of the recombinant bacteria containing the recombinant vector in any of the following aspects:

[0031] (1) application in regulating flood tolerance of plants;

[0032] (2) application in regulating branch number of plants;

[0033] (3) application in regulating pod number per plant;

[0034] (4) application in regulating grain number per plant;

[0035] (5) application in regulating yield per plant;

[0036] (6) application in cultivating flood-tolerant plants.

[0037] Preferably, the GmSUR2 gene is overexpressed to improve flood tolerance, branch number, pod number per plant, grain number per plant and / or yield per plant of the plant. 310

[0038] Preferably, the plant includes Arabidopsis thaliana and soybean.

[0039] The application also provides a method for improving flood tolerance of plants, comprising the following steps: overexpressing the GmSUR2 gene in a recipient plant to improve flood tolerance of the recipient plant; the nucleotide sequence of the GmSUR2 gene is shown as SEQ ID NO. 1. 310 310 The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

[0040] The application also provides a method for improving yield of plants, comprising the following steps: overexpressing the GmSUR2 gene in a recipient plant to improve branch number, pod number per plant, grain number per plant and / or yield per plant of the recipient plant; the nucleotide sequence of the GmSUR2 gene is shown as SEQ ID NO. 1. 310 310 The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

[0041] The application also provides a method for cultivating flood-tolerant transgenic plants, comprising the following steps: overexpressing the GmSUR2 gene in a recipient plant to obtain a flood-tolerant transgenic plant; the nucleotide sequence of the GmSUR2 gene is shown as SEQ ID NO. 1. 310 310 The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

[0042] Preferably, the recipient plant includes Arabidopsis thaliana and soybean. ​​​​

[0043] The present invention discloses the following technical effects:

[0044] This invention overexpresses GmSUR2 in soybean. 310 Gene, obtain GmSUR2 310 Genetically modified soybeans. GmSUR2 310 A comparative analysis of genetically modified soybeans and the genetically modified background material Tianlong No. 1 (TL1) revealed that, compared to TL1, overexpression of GmSUR2... 310 The transgenic lines showed significantly reduced levels of endogenous auxin; after encountering flooding stress, overexpression of GmSUR2 was observed. 310 The transgenic lines showed significantly improved survival rates after flooding stress. Based on field yield statistics, GmSUR2... 310 Genetically modified soybeans showed improved yields, with a significant increase in the number of branches, pods, and grains per plant, as well as the yield per plant.

[0045] This invention also discovered that the transcription factor GmAGL15 can directly bind to GmSUR2. 310 The promoter was inhibited and its expression was suppressed. These results collectively indicate that GmSUR2... 310 This gene plays a crucial role in the synergistic improvement of plant flood tolerance and yield by regulating auxin homeostasis, and is a flood tolerance breeding gene with important application value. Attached Figure Description

[0046] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0047] Figure 1 For GmSUR2 310 AtSUR2 positively regulates plant flood tolerance; a. Statistical analysis of flood tolerance phenotypes and survival rates of different Arabidopsis thaliana genotypes; b. Phylogenetic tree of SUR2 protein; c. GmSUR2 310 Gene expression patterns under flood stress; de. GmSUR2 310 Flood tolerance phenotype and survival rate of transgenic soybeans under greenhouse (d) and field (e) conditions; ** indicates P < 0.01;

[0048] Figure 2 To investigate the negative regulation of auxin-induced plant flood tolerance; a. the effect of flood tolerance stress on the survival rate of different Arabidopsis thaliana lines; b. the effect of salt tolerance treatment on the survival rate of Tianlong No. 1 (TL1) and overexpression of 35S::GmSUR2. 310c. Results of endogenous IAA content detection in soybean plants; d. Effects of exogenous IAA treatment on the flood tolerance of soybean and Arabidopsis thaliana; 1. Results of flood tolerance detection in Arabidopsis thaliana atiaa19 mutant; ** indicates P < 0.01;

[0049] Figure 3 GmAGL15 directly suppresses GmSUR2 310 Expression; a. Yeast one-hybrid validation results; b. Dual-luciferase reporter gene assay results; c. Flood tolerance test results of soybean plants overexpressing GmAGL15; d. GmSUR2 in soybean plants overexpressing GmAGL15. 310 Expression detection results; e. Yeast one-hybrid assay for the detection of GmAGL15 and pHis-GmSUR2 310 Interactions of ABRE motifs in the promoter; pHis-GmSUR2 310 promoter1 (-703 to 0), pHis-GmSUR2 310 promoter2 (-1589 to 0) and pHis-GmSUR2 310 promoter2 mut (ABRE motif ACGTG mutated to AAAAA), three bait constructs, and prey construct pGADT7-GmAGL15 were co-transformed into Y187 yeast strain, and then inoculated onto a specific nutrient-deficient medium; pHis-GmSUR2 310 promoter1 combined with pGADT7, pHis-GmSUR2 310 promoter2 and pGADT7 were used as negative controls for their respective experimental groups; red letters indicate mutated nucleotides; f.EMSA shows GmAGL15 and GmSUR2. 310 ABRE-motif elements in the promoter bind directly; the bound protein-DNA complex and the free probe are indicated by arrows; "+" or "-" indicates the presence (+) or absence (-) of the corresponding component shown on the left; ** indicates P < 0.01;

[0050] Figure 4 For overexpression of 35S::GmSUR2 310 Improvement of soybean field agronomic traits; a. Phenotype of transgenic soybean plants; bg. Phenotypes of transgenic plants in terms of plant height (b), yield per plant (c), number of branches (d), number of pods per plant (e), 100-seed weight (f), number of seeds per plant (g), protein content (h), and oil content (i), respectively; * indicates P < 0.05; ** indicates P < 0.01; ns indicates no significant difference. Detailed Implementation

[0051] The following detailed description of various example embodiments of the application will not be considered to limit the application, but rather to provide a more detailed description of certain aspects, features and embodiments of the application.

[0052] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, for the purposes of the present application, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Furthermore, the use of the terms "about" and "substantially" are understood not to eliminate the effect of the term they modify.

[0053] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the present specification and any document incorporated by reference, the present specification will control.

[0054] Various modifications and changes can be made to the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0055] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional elements or steps.

[0056] Example 1 : GmSUR2 310 Cloning of the gene and construction of overexpression vector

[0057] 1. GmSUR2 310 Amino acid sequence analysis of the protein and GmSUR2 under waterlogging stress 310 Gene expression analysis

[0058] The amino acid sequences of soybean (Glycine max [Williams 82.a2.v1]) and Arabidopsis thaliana [TAIR10] were obtained from the Phytozome v14 database (https: / / phytozome-next.jgi.doe.gov / ). The phylogenetic tree was constructed by MEGA6.06 software using the neighbor-joining method, and 1000 self-exhibition repeated verifications were performed. The results are shown in Figure 1 Fig. 2a, which shows that the AtSUR2 of Arabidopsis thaliana and the GmSUR2 of soybean have the highest homology. 310

[0059] After the soybean variety Williams 82 (W82) was cultivated to the V1 stage, it was subjected to waterlogging stress treatment. The distance from the water surface to the top of the soybean stem was kept at 5 cm, and at 0 days, 1 day, 2 days, 3 days after the start of stress, and 1 day after recovery, leaf tissue samples were collected at each time point, with three independent biological replicates. Total RNA was extracted using a plant RNA extraction kit (Tiangen, Cat. No. DP441, China), cDNA was synthesized according to the manufacturer's instructions (Clontech, Cat. No. 6110A, Japan), and qRT-PCR detection was performed using a SYBR Green mixing kit (Bio-Rad, Hercules, CA, USA) on an ABI 7500 real-time fluorescence quantitative PCR system. The primers used in the quantitative test are as follows:

[0060] β-tubulin-F: 5'-GGAAGGCTTTCTTGCATTGGTA-3' (SEQ ID NO. 6);

[0061] β-tubulin-R: 5'-AGTGGCATCCTGGTACTGC-3' (SEQ ID NO. 7);

[0062] GmSUR2 310 -F: 5'-TCATCCAACACCTTAGAGCCT-3' (SEQ ID NO. 8);

[0063] GmSUR2 310 -R: 5'-GAGAGGTGCCATAGCTGCAT-3' (SEQ ID NO. 9).

[0064] As shown in Figure 1 Fig. 2c, the expression of the GmSUR2 310 gene was reduced under waterlogging stress.

[0065] 2, GmSUR2 310 ​Gene cloning and overexpression vector construction

[0066] Using W82 cDNA as a template, GmSUR2 was obtained by PCR amplification. 310 The complete coding sequence of (Glyma.03G031000) (nucleotide sequence as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.2) was used. The purified PCR product was cloned downstream of the 35S promoter of the plant overexpression vector pCAMBIA1300 using homologous recombination technology to construct the recombinant expression vector 35S::GmSUR2. 310 The cells were then transformed into Agrobacterium GV3101 competent cells and EHA105 competent cells.

[0067] The amplification primers are:

[0068] GmSUR2 310 -OE-F: 5'-TCTGATCAAGAGACAGGATCCATGGTCACAATGCTTTTACCACTTGTTCTA-3' (SEQ ID NO. 10);

[0069] GmSUR2 310 -OE-R: 5'-CGATCGGGGAAAATTCGAGCTCTTATATATGGCTCCTGGTCTTGGCACA-3' (SEQ ID NO. 11).

[0070] Reaction system: KOD FX Neo 0.5 μL, 2×Buffer 12.5 μL, 2mM dNTPs 5 μL, Forwardprimer 1 μL, Reverse primer 1 μL, W82 cDNA 1 μL, dd H2O added to a total volume of 25 μL.

[0071] Reaction procedure: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 0.5 min, 58℃ annealing for 0.5 min, 72℃ extension for 2 min, 35 cycles; 72℃ final extension for 10 min; store at 4℃.

[0072] Example 2: Creation of transgenic plants and identification of flood tolerance phenotypes

[0073] 1. Arabidopsis genetic transformation:

[0074] The Agrobacterium-mediated inflorescence staining method was employed, with the following steps: The correctly constructed vector plasmid 35S::GmSUR2 was... 310The bacteria were activated, suspended in EMS suspension, and activated in the dark for 3 h, and then the bacteria liquid was spotted on the stigma of the flower spikes of the transgenic background material in the flowering stage. The infected Arabidopsis flower spikes were kept moist, and after being cultured in the dark at 21°C for 24 h, they were transferred to a growth environment of 21°C, 16 h light / 8 h dark for further culture until the seeds were harvested, and the positive seedlings were screened. The 35S:: GmSUR2 310 The vector was introduced into the wild type (Col-0) and atsur2 mutant (gene number at4g31500) backgrounds of Arabidopsis thaliana, respectively. After resistance screening and PCR identification, homozygous transgenic lines were obtained.

[0075] The PCR primers, reaction system and reaction procedure for identifying the introduction of the 35S:: GmSUR2310 vector into Arabidopsis thaliana were the same as those used in the construction of the overexpression vector in Example 1 above. Among them, the cDNA in the reaction system was replaced by DNA.

[0076] PCR primers for identifying atsur2 mutant:

[0077] SALK_028573-LP: 5'-TTTCGTGGTTCTCTCTTTTCG-3' (SEQ ID NO. 12);

[0078] SALK_028573-BP: 5'-ATTTTGCCGATTTCGGAAC-3' (SEQ ID NO. 13);

[0079] SALK_028573-RP: 5'-GTGGTATGGGCCATGACTTAC-3' (SEQ ID NO. 14).

[0080] Reaction system: 2x magic Green Taq mix 10 μL, SALK_028573-LP 1 μL, SALK_028573-BP 1 μL, SALK_028573-RP 1 μL, template DNA 1 μL, and dd H2O supplemented to 20 μL.

[0081] Reaction procedure: 95°C pre-denaturation for 10 min; 95°C denaturation for 0.5 min, 56°C annealing for 0.5 min, 72°C extension for 2.5 min, 35 cycles; 72°C final extension for 10 min; 4°C storage.

[0082] 2, Soybean genetic transformation: Taking soybean variety Tianlong No. 1 (TL1) as the transgenic background material, the 35S:: GmSUR2 310The vector was introduced into soybean plants to obtain transgenic soybean plants.

[0083] 3. Submergence tolerance experiment

[0084] The indoor material culture and submergence stress treatment were carried out in a controlled environment greenhouse of Henan Agricultural University (Zhengzhou): Transgenic soybean plants of Tianlong No. 1 (TL1) and overexpressing 35S::GmSUR2 310 were tested, and fully mature seeds were selected, germinated and then transferred to soil. The plants were grown under controlled conditions (25°C, 16h light / 8h dark), and four-week-old soybean plants were subjected to submergence stress treatment, with the water surface kept 5 cm away from the top of the stem, and the submergence lasted for 7 days, followed by 3 days of recovery, and the survival rate was counted.

[0085] The field material culture and submergence stress treatment were carried out in the Yuanyang Experimental Farm of Henan Agricultural University: Transgenic soybean plants of Tianlong No. 1 (TL1) and overexpressing 35S::GmSUR2 310 were tested, and fully mature seeds were selected and sown in the field. Before sowing, the field was carefully leveled to ensure uniform water depth, and to ensure that the material treatment started and ended the stress at the same time. The sowing depth was 3-5 cm, the row spacing was 20 cm, the plant spacing was 5 cm, and the column spacing was 20 cm. The material was cultured for 4 weeks before submergence stress treatment, and the water surface was kept at a water depth of about 5 cm above the stem tip, and the treatment lasted for 7 days, and after 3 days of recovery, the phenotype was observed and the survival rate was counted.

[0086] As shown in a, d, and e of Figure 1 , the results showed that: compared with Tianlong No. 1 (TL1), the survival rate of transgenic Arabidopsis and soybean plants overexpressing GmSUR2 310 after submergence was significantly improved. Overexpression of GmSUR2 310 in atsur2 mutants can complement the submergence tolerance defects. It is indicated that GmSUR2 310 and AtSUR2 positively regulate the submergence tolerance of plants.

[0087] Example 3: Endogenous auxin (IAA) content determination

[0088] The material was cultured in a controlled environment greenhouse of Henan Agricultural University (Zhengzhou), and transgenic soybean plants of Tianlong No. 1 (TL1) and overexpressing 35S::GmSUR2 310 were cultured to V2 stage, and the second trifoliate leaf and stem tip tissues (3 g of fresh sample) were taken, immediately frozen in liquid nitrogen. And ground into fine powder with a pre-cooled mortar, and accurately weighed about 170 mg of sample fine powder. The endogenous hormones were extracted by the methanol method, that is, methanol and 2IAA extraction 24h. LC-MS / MS analysis was performed on a UPLC system (Waters) coupled with a 5500 Qtrap system (AB SCIEX). The chromatographic conditions were as follows: a BEH C18 column (1.7 μm, 100 x 2.1 mm; Waters) was used; mobile phase A: 0.1% (v / v) formic acid in water, mobile phase B: acetonitrile. The elution gradient was as follows: 0-0.5 min, 5% B, 0.5-14 min, 5% B to 25% B; 14-16 min, 25% B to 100% B. The MRM ion pairs of IAA and its internal standard 2 Characteristic ion pairs of IAA. The MRM ion pairs of IAA were m / z 174.1>130.1, 2 The MRM ion pairs of IAA were m / z 176.1>132.1.

[0089] As Figure 2 shown, the results showed that the IAA content in the leaves of transgenic soybean plants overexpressing 35S:: GmSUR2 310 was significantly lower than that of the transgenic background material Tianlong No. 1 (TL1). GmSUR2 310 can complement the flooding tolerance defects of atsur2 mutants.

[0090] Example 4: Functional verification of auxin on flooding tolerance

[0091] (1) Exogenous IAA treatment: 0, 0.25 mg / L and 0.5 mg / L of IAA were added to the flooding treatment solution (ultrapure water), respectively, and Col-0 (4-week-old Col-0 was subjected to flooding stress treatment, with a water surface distance of 5 cm from the Col-0, and the Col-0 was placed in complete darkness for 2 days of flooding treatment, followed by 7 days of recovery growth, and the survival rate was counted) and Tianlong No. 1 (TL1) [the culture and flooding stress treatment of indoor materials were carried out in a controlled environment greenhouse of Henan Agricultural University (Zhengzhou): TL1 and transgenic soybean overexpressing 35S:: GmSUR2 310 were tested, and fully matured seeds were selected, germinated and transferred to soil; the plants were grown under controlled conditions (25°C, 16 hours of light / darkness 8 hours), and four-week-old soybean plants were subjected to flooding stress treatment, with a water surface distance of 5 cm from the soybean shoot tip, and the flooding lasted for 7 days, followed by 3 days of recovery growth, and the survival rate was counted] were subjected to flooding stress treatment.

[0092] (2) Mutant treatment: the auxin signal-deficient mutant atiaa19 (AT3G15540) of Arabidopsis thaliana was subjected to flooding stress treatment.

[0093] As Figure 2As shown in the results, the concentration of exogenous IAA treatment was negatively correlated with the flooding tolerance of Arabidopsis and soybean. The survival rate of atiaa19 mutant was higher than that of Col-0 after flooding stress, indicating that the atiaa19 mutant had stronger flooding tolerance.

[0094] Example 5: Transcription factor GmAGL15 inhibits the transcription of GmSUR2 310

[0095] 1. Yeast one-hybrid experiment: 5 μg of two pre-cooled plasmids GmSUR2 310 prompter-pHis and GmAGL15-AD, 10 μL of salmon sperm pre-denatured at 95°C for 5 min, were added to Y187 yeast competent cells, mixed with 500 μL of PEG / LiAc. The transformation was completed after 30°C water bath for 30 min and 42°C water bath for 15 min. The transformed product was plated on SD / -Leu / -Trp selection medium, and after 48-96 h of culture at 28°C, single colony was plated on SD / -Leu / -Trp / His selection medium containing 150 mM 3-AT (3-amino-1,2,4-triazole) to verify the interaction.

[0096] As Figure 3 shown, the experimental group grew normally on the selection medium, while the combination of pGADT7 empty vector and GmAGL15-AD could not grow, indicating that GmSUR2 310 prompter (nucleotide sequence as shown in SEQ ID NO. 3) and GmAGL15 (nucleotide sequence as shown in SEQ ID NO. 4, protein sequence as shown in SEQ ID NO. 5) combined.

[0097] 2. Gel retardation (EMSA)

[0098] Prokaryotic expression and purification of MBP-GmAGL15 fusion protein, synthesis of biotin-labeled wild-type and mutant GmSUR2 310 promoter probe, binding reaction, and detection of protein-DNA complex. The specific steps are as follows:

[0099] (1) Prokaryotic expression and purification of MBP-GmAGL15 fusion protein, wherein the primers for constructing the prokaryotic expression vector are as follows:

[0100] GmAGL15-MBP-F: 5'-AACCTCGGGCACCATCACCATATGGGTCGAGGGAAAATCGAG-3' (SEQ ID NO. 15);

[0101] ​GmAGL15-MBP-R: 5'-CTGCAAGGCGATTAAGTTGGTCATTTGAAAAGGTTTCTTTCTTGGGG-3' (SEQ ID NO. 16).

[0102] The MBP-GmAGL15 plasmid with the vector connected was transformed into BL21 competent cells, and positive colonies were shaken in 150 mL of liquid LB medium. When the bacterial liquid concentration OD 600 When the OD reached 0.6-0.8, 1 mM IPTG was added, and the bacteria were collected after being induced at 16°C, 120 rpm for 16 h. Then the bacteria were suspended with 1×PBS, and then 1 mM PMSF and 1 mg / mL lysozyme were added for lysis on ice for 30 min. After lysis, the ultrasonic disrupter power was set to 250 w, and the working time was 7 s and the stop time was 10. After 30 min of crushing, the supernatant was obtained by centrifugation at 4°C, 12000 rpm for 30 min. The target protein was purified using the Biyun Tian P2226 HIS tag protein purification kit (reduction-resistant chelating type) for subsequent experiments.

[0103] (2) Synthesis of biotin-labeled wild-type and mutant GmSUR2 310 The promoter probe is as follows:

[0104] The probe design is shown in Table 1.

[0105] Table 1 Probe sequence

[0106]

[0107] Probe synthesis: according to the identification of the primer tube, add ddH2O, take equal volume of forward and reverse primers for annealing experiment, in the PCR instrument, 95°C, 2 min, gradually cool to room temperature. Dilute the labeled probe to 100 nmol / L, and store at -20°C for standby.

[0108] Protein and probe combination: add the following reagents in the following order: water, 5×binding buffer, protein (2 μg), probe. When doing cold competition, first add unlabeled probe (10×, 50×, 100× of labeled probe, respectively), mix well, then incubate at room temperature for 10 min, then add labeled probe, mix gently, incubate in the PCR instrument (25°C) for 30 min. Then add 2 μL of 6×EMSA / Gel-Shift loading buffer (blue) for gel running. The addition of components and the amount are shown in Table 2.

[0109] Table 2 System setting

[0110]

[0111] (3) Dual-luciferase reporter assay

[0112] In N. benthamiana leaves, 35S::GmAGL1-GFP and Pro-GmSUR2 310 ::LUC were co-injected, and luciferase activity was detected.

[0113] The 35S::GmAGL1-GFP vector construction primers are as follows:

[0114] GmAGL15-GFP-F: 5'-TCTGATCAAGAGACAGGATCCATGGGTCGAGGGAAAATCGAG-3' (SEQ ID NO. 21);

[0115] GmAGL15-GFP-R: 5'-GCCCTTGCTCACCATGGATCCTCATTTGAAAAGGTTTCTTTCTTGGGG-3' (SEQ ID NO. 22).

[0116] The reaction system was as follows: KOD FX Neo 0.5 μL, 2x Buffer 12.5 μL, 2 mM dNTPs 5 μL, GmAGL15-GFP-F 1 μL, GmAGL15-GFP-R 1 μL, soybean DNA 1 μL, and dd H2O supplemented to 25 μL.

[0117] The reaction procedure was as follows: 95°C pre-denaturation for 10 min; 95°C denaturation for 0.5 min, 58°C annealing for 0.5 min, 72°C extension for 1 min, 35 cycles; 72°C final extension for 10 min; and 4°C storage.

[0118] Pro-GmSUR2 310 The Pro-GmSUR2::LUC vector construction primers are as follows:

[0119] GmSUR2 310 -pGREENII 0800-F: 5'-CTATAGGGCGAATTGGGTACCATTCTTTTGAGTTTTGAATCCGAC-3' (SEQ ID NO. 23);

[0120] GmSUR2 310 -pGREENII 0800-R: 5'-CTCGAGGGGGGGCCCGGTACCGAGGAAGAAAAAAGTTGTAAATCA-3' (SEQ ID NO. 24).

[0121] The reaction system is: KOD FX Neo 0.5 μL, 2x Buffer 12.5 μL, 2mM dNTPs 5 μL, GmSUR2 310 -pGREENII 0800-F 1 μL, GmSUR2 310 -pGREENII 0800-R 1 μL, W82 DNA 1 μL, ddH2O supplemented to 25 μL.

[0122] The reaction procedure is: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 0.5 min, 58℃ annealing for 0.5 min, 72℃ extension for 2.5 min, 35 cycles; 72℃ final extension for 10 min; 4℃ storage.

[0123] The constructed vector plasmid is respectively transferred into GV3101 psoup p19 competent cells, and positive clones are selected for activation. After 12 h of activation in 150 mL liquid LB medium at 28℃ and 220 rpm on a shaker, the bacterial liquid is enriched, the precipitate is suspended with MES suspension (10 mM MgCl2, 10 mM MES, 200 μM AS), and the concentration of the bacterial liquid is adjusted to OD 600 =1.0, the experimental group is GmSUR2 310 -pGREENII 0800+ GmAGL15-GFP, and the control group is GmSUR2 310 -pGREENII 0800+GFP, the two bacterial liquids are mixed at a ratio of 1:1, and after 3 h of activation at 28℃ in the dark, injection is performed, and each combination is injected in the size of a coin. After 12 h of cultivation of the tobacco in the dark at 25℃, the tobacco is transferred to an environment of 25℃ 16 h light / 8 h dark for continued cultivation, and after 36-48 h from the injection time, the back of the injected tobacco is smeared with potassium salt of fluorescein, and then observed using a live imaging instrument.

[0124] As Figure 3 shown, the dual luciferase experiment proves that GmAGL15 significantly inhibits the activity of the GmSUR2 310 promoter, the GmSUR2 310 expression in the soybean plant overexpressing GmAGL15 is down-regulated, and the flood tolerance of the soybean plant overexpressing GmAGL15 is reduced. The yeast one-hybrid and gel retardation experiments prove that GmAGL15 can directly bind to the ABRE motif on the GmSUR2 310 promoter.

[0125] Example 6: Evaluation of field agronomic traits of transgenic soybeans

[0126] Under normal field conditions, Tianlong No. 1 (TL1) and overexpression 35S:: GmSUR2310 The homozygous transgenic soybean lines were studied. Plant height, number of branches per plant, number of pods per plant, number of grains per plant, 100-grain weight, and yield per plant were assessed at maturity.

[0127] like Figure 4 As shown, the results indicate that, compared with Tianlong-1 (TL1), overexpression of 35S::GmSUR2 310 The transgenic lines showed a significant increase in the number of branches per plant, the number of pods per plant, the number of seeds per plant, and the yield per plant, while there were no significant differences in 100-seed weight, plant height, protein content, and oil content.

[0128] The gene protein sequence involved in this invention is:

[0129] GmSUR2 310 The nucleic acid sequence (SEQ ID NO.1) is as follows:

[0130]

[0131] GmSUR2 310 The encoded protein sequence (SEQ ID NO. 2) is:

[0132] MVTMLLPLVLCLTLPVFFLFFIQHLRAFKKPPLPPGPKGLPIIGNLHKLDNSILCMQLWHLSKKYGPIFSLQLGLRKTIVISSPKLAKEVLKNHDLEFSGRPKLLPQQKLSYNGSEIVFSPYNEYWREMRKICVAHIFSSKRVSSFSSIRKFEVKQMIKTISGHASSSGVTNLSELLISLSSTIICRVAFGRRYEDEGSERSRFHGLLNELQVLMGTFFISDFIPFTGWIDKLKGLHARLERNFKELDKFYQEVIDEHMDPNRQHAEEQDMVDVLLQLKNDRSLSIDLTYDHIKGVLMNILAAGTDTTAATSVWAMTALVKNPRVMKKVQEEVRNVGGTKDFLDEDDIQKLPYFKAMIKETLRLHLPGPLLVPRESTEECIVDGYRIPAKTIVYVNAWVIQRDPEVWKNPEEFCPERFLDSAIDYRGQDFELIPFGAGRRICPGILMAAVTLELVLANLLHSFDWELPQGIVKEDIDFEVLPGITQHKKNHLCLCAKTRSHI.

[0133] GmSUR2 310 The prompter sequence (Glyma.03G031000, SEQ ID NO. 3) is:

[0134]

[0135] GmAGL15 CDS region sequence (Glyma.11G158812, SEQ ID NO. 4):

[0136] ATGGGTCGAGGGAAAATCGAGATCAAAAGAATCGACAATGCTAGCAGCAGACAAGTCACGTTCTCGAAGCGGAGAACAGGGTTGTTCAAGAAGGCTCAGGAACTTTCCATTCTCTGTGACGCCGAGGTTGCTGTCATAGTTTTCTCCAACACTGGCAAGCTCTTCGAGTTTTCCAGTTCCGGTATGAAGCGAACACTTTCAAGATACAACAAATGCCTTGGTTCTACAGATGCTGCTGTAGCAGAAATTATGACACAGAAGGAAGATTCTAAGATGGTGGAGATTCTAAGAGAGGAAATTGAAAAGCTAGAAACAAAGCAATTACAGTTGGTGGGTAAGGATCTGACAGGATTGGGTTTAAAGGAATTGCAAAATTTAGAGCAGCAACTTAATGAGGGGTTATTGTCTGTCAAGGCGAGAAAGGAGGAATTACTCATGGAGCAACTAGAGCAATCTAGAGTTCAGGAACAGCGGGTTATGTTGGAGAATGAAACTTTGCGAAGACAGATTGAGGAGCTTCGGTGTCTGTTTCCACAATCAGAAAGCATGGTCCCATTCCAATACCAACATACTGAAAGAAAGAATACTTTTGTAAATACTGGCGCCAGATGTCTCAACTTGGCTAATAACTGTGGAAATGAGAAAGGGAGTTCAGATACAGCATTTCATTTGGGGTTGCCTGCTGGTGTTCAAGAGGAAGGCCCCCAAGAAAGAAACCTTTTCAAATGA.

[0137] > GmAGL15 (Glyma.11g158812.1.p protein sequence, SEQ ID NO. 5):

[0138] MGRGKIEIKRIDNASSRQVTFSKRRTGLFKKAQELSILCDAEVAVIVFSNTGKLFEFSSSGMKRTLSRYNKCLGSTDAAVAEIMTQKEDSKMVEILREEIEKLETKQLQLVGKDLTGLGLKELQNLEQQLNEGLLSVKARKEELLMEQLEQSRVQEQRVMLENETLRRQIEELRCLFPQSESMVPFQYQHTERKNTFVNTGARCLNLANNCGNEKGSSDTAFHLGLPAGVQEEGPQERNLFK.

[0139] The above-described embodiments are merely preferred ways of implementing the present application, and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the scope of protection of the present application as defined by the claims.

Claims

1. GmSUR2 310 use of the gene in any of the following: (1) use in modulating flood tolerance of a plant; (2) use in modulating branch number of a plant; (3) use in modulating pod number per plant of a plant; (4) use in modulating grain number per plant of a plant; (5) use in modulating yield per plant of a plant; (6) use in breeding a flood-tolerant plant; wherein The GmSUR2 310 The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

2. GmSUR2 310 Proteins of gene expression for use in any of the following: (1) use in modulating flood tolerance of a plant; (2) use in modulating branch number of a plant; (3) use in modulating pod number per plant of a plant; (4) use in modulating grain number per plant of a plant; (5) use in modulating yield per plant of a plant; (6) use in breeding a flood-tolerant plant; wherein The GmSUR2 310 The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence of the protein is shown as SEQ ID NO.

2.

3. A recombinant vector comprising the GmSUR2 310 gene for use in any one of: (1) use in modulating flood tolerance of a plant; (2) use in modulating branch number of a plant; (3) use in modulating pod number per plant of a plant; (4) use in modulating grain number per plant of a plant; (5) use in modulating yield per plant of a plant; (6) use in breeding a flood-tolerant plant; wherein, The recombinant vector is constructed by introducing the GmSUR2 310 gene into an expression vector. 310 The nucleotide sequence of the GmSUR2 gene is shown as SEQ ID NO.

1.

4. Use of a recombinant bacterium comprising the recombinant vector of claim 3 in any one of: (1) modulating flood tolerance of a plant; (2) modulating branch number of a plant; (3) modulating pod number per plant of a plant; (4) modulating grain number per plant of a plant; (5) modulating yield per plant of a plant; (6) breeding a flood-tolerant plant.

5. Use according to any one of claims 1 to 4, wherein the compound is of formula (I) ###0002### (I) or a pharmaceutically acceptable salt thereof. Overexpression of said GmSUR2 310 increases the flood tolerance, the number of branches, the number of pods per plant, the number of seeds per plant and / or the yield per plant.

6. Use according to any one of claims 1 to 4, wherein The plant includes Arabidopsis thaliana and soybean.

7. A method of increasing the flood tolerance of a plant, comprising introducing into said plant a nucleic acid molecule encoding a polypeptide having the amino acid sequence of SEQ ID NO:

2. comprising the following steps: Overexpressing GmSUR2 in a recipient plant 310 gene to improve the flooding tolerance of the recipient plant; the GmSUR2 310 The nucleotide sequence of the GmSUR2 gene is shown as SEQ ID NO.

1.

8. A method of increasing yield in a plant, comprising, comprising the following steps: Overexpressing GmSUR2 in a recipient plant 310 to increase the number of branches, the number of pods per plant, the number of seeds per plant and / or the yield per plant of said recipient plant; the nucleotide sequence of the GmSUR2 310 gene is shown as SEQ ID NO.

1.

9. A method of breeding a transgenic plant for submergence tolerance, comprising, comprising the following steps: Overexpression of GmSUR2 in the recipient plants 310 gene, to obtain a submergence tolerance transgenic plant; the nucleotide sequence of the GmSUR2 310 gene is shown as SEQ ID NO.

1.

10. The method according to any one of claims 7 to 9, characterized in that, The recipient plant includes Arabidopsis thaliana and soybean.

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