Soybean GmERE1 gene and application thereof in drought and salt stress resistance

Abstract

The application provides application of a soybean GmERE1 gene in drought resistance and salt stress resistance, and belongs to the technical field of plant genetic engineering.The nucleotide sequence of the GmERE1 gene is shown as SEQ ID NO.1, and the protein sequence coded by the GmERE1 gene is shown as SEQ ID NO.2.The application also provides a recombinant vector for overexpressing and knocking down GmERE1 gene expression, and the recombinant vector is transformed into Agrobacterium rhizogenes;transgenic complex plants are obtained by using the obtained Agrobacterium rhizogenes to infect seeds, so that overexpression and knockdown expression of the GmERE1 gene are realized.Combination of GmERF5 and GmERE1 can improve the activity of peroxidase in soybean plants by promoting the expression of GmPOD1, and then improve the drought resistance and salt tolerance of the plants.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and in particular relates to a soybean GmERE1 gene and its application in drought resistance and salt stress tolerance. Background Technology

[0002] Soybean (Glycine max L.) is an important food and economic crop. Soybeans have a poorly developed root system and require a large amount of water during their growth and development, making them the most water-sensitive legume. Therefore, soybeans are frequently affected by adverse environmental factors such as drought and high salinity in the field: leaf wilting, yellowing, curling, and stunted growth are common, thus affecting yield and quality, and in severe cases, leading to plant death. Therefore, identifying drought- and salt-tolerant genes, studying the molecular mechanisms of soybean drought and salt tolerance, breeding drought- and salt-tolerant soybean varieties using genetic engineering methods, and elucidating the response mechanisms of soybeans to abiotic stresses are of great significance for improving soybean yield.

[0003] AP2 / ERF transcription factors play important roles in plant stress responses, particularly in drought and salt stress. ERF-TFs are widely involved in the response processes to abiotic stresses such as drought and salt in many species. GmERF3, TSRF1, OsERF48, and TaERF3 have been identified as positive regulators of drought resistance in soybean, rice, and wheat, respectively. In Arabidopsis, the AP2 / ERF transcription factor TINY inhibits brassinosteroids (BR) in regulating plant growth, and actively regulates drought response by activating drought-responsive genes and promoting abscisic acid-mediated stomatal closure. TaERF3 actively regulates wheat responses to salt and drought stress by activating stress-related genes. In apples subjected to drought stress, MdERF38 enhances anthocyanin biosynthesis by increasing the transcriptional activity of MdMYB1, thereby enhancing drought resistance. In rice, overexpression of OsAP37 enhances drought tolerance and increases grain yield. Under stress conditions, overexpression of OsEREBP1 activates the JA and ABA signaling pathways, thereby improving rice survival. OsERF71 activates various stress response, cell wall-related, and lignin biosynthesis-related genes, leading to changes in root structure, increased aerenchyma, and radial root growth, thus improving the drought resistance of rice shoots. Several ERFs in wheat have been identified, and their regulation may be used for variety improvement. TaERF3 enhances tolerance to salt and drought stress; overexpression of TaERF3 increases proline and chlorophyll content in transgenic lines and inhibits H2O2 formation and stomatal conductance. Overexpression of AtERF019 delays flowering, plant growth, and senescence in Arabidopsis, thus producing drought resistance. AtERF96 has been shown to respond to ABA signaling; its overexpression significantly increases the expression levels of ABA-responsive genes, and AtERF96 overexpression exhibits an ABA-sensitive response with reduced stomatal diameter.

[0004] To date, only a small number of quantitative trait loci (QTLs) associated with drought and salt stress in soybean have been identified, and the number of genes involved in the regulation of drought and salt stress in soybean is limited, with their regulatory networks remaining unclear. Therefore, research on the regulatory genes of drought and salt stress in soybean is of great significance. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a soybean GmERE1 gene and its application in drought resistance and salt stress tolerance.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a GmERE1 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The present invention also provides a protein encoded by the GmERE1 gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] The present invention also provides a recombinant vector for overexpressing the GmERE1 gene, the recombinant vector comprising an initial expression vector and the GmERE1 gene.

[0010] Preferably, the initial expression vector is the pCAMBIA3301-GFP vector.

[0011] The present invention also provides a recombinant vector for knocking down the expression of the GmERE1 gene, the recombinant vector comprising the pFGC5941 vector and the GmERE1 gene.

[0012] The present invention also provides an application of the GmERE1 gene in altering the drought resistance and / or salt tolerance of plants, wherein overexpression of the GmERE1 gene in plants reduces the drought resistance and / or salt tolerance of plants; and knockdown of the GmERE1 gene expression in plants increases the drought resistance and / or salt tolerance of plants.

[0013] Preferably, the method for overexpressing the GmERE1 gene includes the following steps:

[0014] The GmERE1 gene was cloned into the pCAMBIA3301-GFP vector to obtain the recombinant vector;

[0015] The obtained recombinant vector was transformed into Agrobacterium rhizogenes, and the transformed Agrobacterium rhizogenes was used to infect seeds to obtain transgenic complex plants, thereby achieving overexpression of the GmERE1 gene.

[0016] Preferably, the method for knocking down GmERE1 gene expression includes the following steps:

[0017] The GmERE1 gene was cloned into the pFGC5941 vector to obtain the recombinant vector;

[0018] The obtained recombinant vector was transformed into Agrobacterium rhizogenes, and the transformed Agrobacterium rhizogenes was used to infect seeds to obtain transgenic complex plants, thereby achieving the knockdown expression of the GmERE1 gene.

[0019] This invention also provides an application of the GmERE1 gene as a target in improving crop drought and salt tolerance. The GmERF5 gene improves the drought resistance and salt tolerance of plants by negatively regulating the GmERE1 gene.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. Overexpression of GmERE1 in this invention can inhibit antioxidant enzyme activity, thereby reducing the drought resistance and salt tolerance of transgenic complex plants. The GmERE1 gene can specifically bind to the DRE element on the GmPOD1 promoter, and can directly regulate and inhibit the expression of GmPOD1.

[0022] 2. The GmERF5 gene described in this invention can specifically bind to the GCC-box element on the GmERE1 promoter, directly regulating and inhibiting GmERE1 expression. Therefore, the combination of GmERF5 and GmERE1 can enhance the expression of GmPOD1, thereby increasing the activity of peroxidase in soybean plants and thus improving the drought and salt tolerance of the plants.

[0023] 3. In this invention, the pCAMBIA3301 vector is modified by inserting GFP protein, and transgenic hairy roots can be identified by GFP fluorescence.

[0024] 4. The soybean GmERF5 and GmERE1 of this invention provide a new regulatory gene resource for improving the drought resistance and salt tolerance of soybeans. They can be used for the breeding and improvement of stress-resistant soybean materials, laying a theoretical foundation for the molecular mechanism of drought resistance and salt tolerance, and also providing a theoretical basis and gene resource for the molecular breeding of soybeans for drought resistance and salt tolerance. Attached Figure Description

[0025] Figure 1 This is a graph showing the GFP detection results of hairy roots of GmERE1-OE transgenic soybean obtained by GmERE1 overexpression;

[0026] Figure 2 These are the results of Western blot analysis of hairy roots from GmERE1-OE transgenic soybean obtained by GmERE1 overexpression.

[0027] Figure 3 This is a rapid detection result of Bar protein in the hairy roots of GmERE1-RNAi transgenic soybean obtained by knocking down GmERE1 gene expression;

[0028] Figure 4 The graph shows the relative expression levels of GmERE1 in the hairy roots of GmERE1-OE transgenic soybean obtained by overexpressing GmERE1 and GmERE1-RNAi transgenic soybean obtained by knocking down GmERE1 gene expression.

[0029] Figure 5 The leaf growth of GmERE1-OE transgenic soybean complex plants and GmERE1-RNAi transgenic soybean complex plants under drought conditions.

[0030] Figure 6These are images of leaf stomata measured using ViewPoint software from GmERE1-OE and GmERE1-RNAi transgenic soybean complex plants under drought conditions.

[0031] Figure 7 The ratio of stomatal width to height in leaves of GmERE1-OE transgenic soybean complex plants and GmERE1-RNAi transgenic soybean complex plants under drought conditions.

[0032] Figure 8 The graphs show the SOD activity of GmERE1-OE and GmERE1-RNAi transgenic soybean complex plants under drought conditions.

[0033] Figure 9 These are POD activity diagrams of GmERE1-OE transgenic soybean complex plants and GmERE1-RNAi transgenic soybean complex plants under drought conditions.

[0034] Figure 10 This is a graph showing the SOD activity of GmERE1-OE transgenic soybean complex plants and GmERE1-RNAi transgenic soybean complex plants under 250mM salt treatment.

[0035] Figure 11 This is a graph showing the POD activity of GmERE1-OE transgenic soybean complex plants and GmERE1-RNAi transgenic soybean complex plants under 250mM salt treatment.

[0036] Figure 12 These are the LUC / REN relative activity assay results for p35S / pDRE:LUC and p35S:GmERE1 / pDRE:LUC;

[0037] Figure 13 The enrichment of GmERE1 protein and the P1 region of the GmPOD1 promoter in the hairy roots of GmERE1-OE transgenic plants;

[0038] Figure 14 Analysis of GmPOD1 expression levels in the hairy roots of GmERE1 transgenic plants under drought and salt stress;

[0039] Figure 15 The images show the detection results of p35S / pGCC:LUC and p35S:GmERF5 / pGCC:LUC (where a is the chemiluminescence imaging of p35S / pGCC:LUC and p35S:GmERF5 / pGCC:LUC, and b is the LUC / REN relative activity detection result of p35S / pGCC:LUC and p35S:GmERF5 / pGCC:LUC).

[0040] Figure 16 The images show the detection results for p35S / pGmERE1:LUC and p35S:GmERF5-myc / pGmERE1:LUC (where a is the chemiluminescence imaging of p35S / pGmERE1:LUC and p35S:GmERF5-myc / pGmERE1:LUC, and b is the LUC / REN relative activity detection result of p35S / pGmERE1:LUC and p35S:GmERF5-myc / pGmERE1:LUC). Detailed Implementation

[0041] This invention provides a GmERE1 gene, the nucleotide sequence of which is shown in SEQ ID NO.1, specifically as follows:

[0042] .

[0043] This invention also provides a protein encoded by the GmERE1 gene, the amino acid sequence of which is shown in SEQ ID NO.2, specifically:

[0044] MVKPKSVEKPAEEQQQRSVSSYRGVRKRKWGKYVSEIRLPNSRQRIWLGSYDSAEKAARAFDAAMFCLRGSGAKFNFPSDPPNIAGGGNMTSSQIQIAAARFANSEPRNERSDQPVESLTSDEETASFPVISDTDTSSPPLSDVTFQNDAELVTGSFPDMFSDFGSGDFVPDFSDFPSFDDFSRDFFLHELPGFNFGEENLDGLIIQDSFLWNF.

[0045] The present invention also provides a recombinant vector for overexpressing the GmERE1 gene, the recombinant vector comprising an initial expression vector and the GmERE1 gene.

[0046] Preferably, the initial expression vector is the pCAMBIA3301-GFP vector (http: / / www.biofeng.com / ). In this invention, the recombinant vector is constructed by ligating the GmERE1 gene into the pMDTM18-T vector, followed by transformation, identification, and extraction. The extracted recombinant plasmid is then identified and double-digested with the pCAMBIA3301-GFP vector plasmid to obtain a recombinant vector overexpressing the GmERE1 gene.

[0047] This invention also provides a recombinant vector for knocking down the expression of the GmERE1 gene, the recombinant vector comprising the pFGC5941 vector (http: / / www.biofeng.com / ) and the GmERE1 gene. The recombinant vector is constructed by using the GmERE1 sequence as a template for homology comparison to find its homologous gene in soybean, designing specific primers to amplify the dissimilar portion of GmERE1 from its homologous gene via PCR, and then ligating the amplified fragment forward and reverse into the plant expression vector pFGC5941, respectively, to create a hairpin structure in the transcription region.

[0048] The present invention also provides an application of the GmERE1 gene in altering the drought resistance and / or salt tolerance of plants, wherein overexpression of the GmERE1 gene in plants reduces the drought resistance and / or salt tolerance of plants; and knockdown of the GmERE1 gene expression in plants increases the drought resistance and / or salt tolerance of plants.

[0049] Preferably, the method for overexpressing the GmERE1 gene includes the following steps:

[0050] The GmERE1 gene was cloned into the pCAMBIA3301-GFP vector to obtain the recombinant vector;

[0051] The obtained recombinant vector was transformed into Agrobacterium rhizogenes, and the transformed Agrobacterium rhizogenes was used to infect seeds to obtain transgenic complex plants, thereby achieving overexpression of the GmERE1 gene.

[0052] In this invention, the method of transforming into Agrobacterium rhizogenes is preferably to add the recombinant vector plasmid into K599 Agrobacterium rhizogenes competent cells, wherein the volume ratio of the recombinant vector plasmid to K599 Agrobacterium rhizogenes competent cells is preferably 1:5-15, more preferably 1:8-12; and the Agrobacterium rhizogenes is activated before infection.

[0053] Preferably, the method for knocking down GmERE1 gene expression includes the following steps:

[0054] The GmERE1 gene was cloned into the pFGC5941 vector to obtain the recombinant vector;

[0055] The obtained recombinant vector was transformed into Agrobacterium rhizogenes, and the transformed Agrobacterium rhizogenes was used to infect seeds to obtain transgenic complex plants, thereby achieving the knockdown expression of the GmERE1 gene.

[0056] In this invention, the method of transforming into Agrobacterium rhizogenes is preferably to add the recombinant vector plasmid into K599 Agrobacterium rhizogenes competent cells; the volume ratio of the recombinant vector plasmid to K599 Agrobacterium rhizogenes competent cells is preferably 1:5-15, more preferably 1:8-12; and Agrobacterium rhizogenes is activated before infection.

[0057] This invention also provides an application of the GmERE1 gene as a target in improving crop drought and salt tolerance. The GmERF5 gene improves the drought resistance and salt tolerance of plants by negatively regulating the GmERE1 gene.

[0058] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0059] Example 1

[0060] Construction of the plant overexpression vector pCAMBIA3301-GmERE1-GFP:

[0061] (1) Primer design

[0062] The overexpression recombinant vector pCAMBIA3301-GmERE1-GFP was constructed. Using cDNA from the soybean cultivar "Dongnong 50" as a template, specific primers were designed, and RT-PCR amplification was performed to obtain the full-length GmERE1 fragment. This fragment was then ligated into the pMDTM18-T vector. The recombinant plasmid and pCAMBIA3301-GFP plasmid, which had been confirmed by sequencing, were digested with enzymes at Nco1 and BglII. The digested product containing the GmERE1 fragment was then ligated into pCAMBIA3301-GFP to obtain the pCAMBIA3301-GmERE1-GFP recombinant vector.

[0063] The primer sequences are as follows:

[0064] F(SEQ ID NO.3):

[0065] CCATGGTTATGGTGAAGCCGAAGAGCG

[0066] R(SEQ ID NO.4):

[0067] AGATCTGAAATTCCACAAGAACGAGTCCT

[0068] (2) Linkage reaction of T-carrier

[0069] The target fragment was ligated into the pMDTM18-T vector, and the ligation reaction system was as follows:

[0070] Solution I: 4μL

[0071] pMDTM18-T: 1μL

[0072] Gel recovery product: 5μL

[0073] Total volume: 10μL

[0074] After thorough mixing, react at 16°C for 4 hours.

[0075] (3) Conversion of the linker products

[0076] 1) Thaw E. coli DH5α competent cells on ice.

[0077] 2) Add the product obtained in step (2) to E. coli DH5α competent cells and incubate on ice for 30 min.

[0078] 3) After the ice bath, quickly transfer the device to a 42°C temperature for 42 seconds, then quickly transfer it again and ice bath for 3 minutes.

[0079] 4) Add 700 μL of LB liquid, place in a shaker, and incubate at 37°C and 220 rpm for 60 min.

[0080] 5) Take 200 μL of the bacterial suspension and spread it evenly on a screening plate. Incubate at 37°C upside down overnight.

[0081] (4) PCR identification of transformed clones

[0082] 1) Select plump single spots and culture them in liquid screening medium at 37℃ and 220rpm for 4-6 hours.

[0083] 2) Bacterial culture PCR identification was performed using Mix enzymes. The PCR system is as follows:

[0084] 2×Taq Master Mix enzyme: 12.5μL

[0085] Bacterial solution: 1 μL

[0086] Primer F (10 μM): 1 μL

[0087] Primer R (10 μM): 1 μL

[0088] ddH2O: 9.5 μL

[0089] Total volume: 25μL

[0090] 3) Amplify on a PCR instrument: 98℃ for 3 min; 98℃ for 30 sec, 60℃ for 30 sec, 72℃ for 60 sec, repeat 30 times; 72℃ for 7 min; store at 4℃.

[0091] 4) Detect the PCR amplification products by electrophoresis, select the bacterial solutions that are identified as positive, and use them for plasmid extraction.

[0092] (5) Extraction of recombinant plasmids

[0093] 1) Take 2 mL of bacterial culture and centrifuge at 12000 rpm for 1 min.

[0094] 2) Add 250 μL of SI liquid to resuspend the bacterial pellet.

[0095] 3) Add 250 μL of SⅡ liquid and shake well several times.

[0096] 4) Add 350 μL of SⅢ liquid, shake well, and centrifuge at 12000 rpm for 10 min.

[0097] 5) Pipette 800 μL of supernatant into the adsorption column, centrifuge at 12000 rpm for 1 min, and remove the waste liquid at the bottom of the tube.

[0098] 6) Add 500 μL of WI solution, centrifuge at 12000 rpm for 1 min, and remove the waste liquid at the bottom of the tube.

[0099] 7) Add 700 μL of WII solution, centrifuge at 12000 rpm for 1 min, and remove the waste liquid at the bottom of the tube. Repeat once.

[0100] 8) Place the preparation tube back into the EP tube and centrifuge for 1 min.

[0101] 9) Place the preparation tube into a new EP tube, add 30 μL of Elution Buffer, centrifuge at 12000 rpm for 1 min, and store at -20℃.

[0102] (6) Double enzyme digestion identification of recombinant plasmids

[0103] The double enzyme digestion system is as follows:

[0104] Recombinant plasmid: 16 μL

[0105] 10×Buffer: 2μL

[0106] Endonuclease I: 1 μL

[0107] Endonuclease II: 1 μL

[0108] Total volume: 20μL

[0109] React at 37℃ for 30 min, then perform 1% agarose gel electrophoresis for detection.

[0110] (7) Sequencing

[0111] The plasmid was correctly digested with double enzymes and then sequenced for identification. The bacterial culture with correct sequencing results was mixed with 30% glycerol at a 1:1 ratio to preserve the bacterial strain.

[0112] (8) Construction of the carrier

[0113] 1) Perform double digestion of the recombinant plasmid that has been sequenced without error and the pCAMBIA3301-GFP plasmid, respectively. Refer to step (6) for the system and reaction conditions.

[0114] 2) Recover the target fragment and the digested vector fragment according to the instructions of the OMEGA gel recovery kit.

[0115] 3) The ligation of the target gene DNA fragment with the pCAMBIA3301-GFP vector fragment is performed using the following specific system:

[0116] Target gene DNA fragment: 2μL

[0117] DNA fragment of the vector: 2 μL

[0118] 10×T4 DNALigase Buffer: 1μL

[0119] T4 DNALigase: 0.5 μL

[0120] ddH2O: 4.5μL

[0121] Total volume: 10μL

[0122] Reaction conditions: 22℃, 120min.

[0123] 4) Transform the ligation product into E. coli using E. coli DH5α competent cells, following the method in step (3).

[0124] 5) PCR detection, the method is the same as step (4).

[0125] 6) The positive recombinant plasmids obtained by detection are identified by double enzyme digestion, and the method is the same as in step (6).

[0126] Example 2

[0127] Construction of a recombinant vector to knock down GmERE1 gene expression:

[0128] 1. Primer design

[0129] GmERE1-RNAi1F (SEQ ID NO.5):TTTCATTTGGAGAGGACACGCTCGAGTGTTTCATCGTACAGAGGAGT

[0130] GmERE1-RNAi1R (SEQ ID NO.6): AATCATCGATTGGGCGCGCCCCATGGCTGAATCTGGGAGGAGGTC

[0131] GmERE1-RNAi2F (SEQ ID NO.7):TCCCGGGTCTTAATTAACTCTCTAGATGTTTCATCGTACAGAGGAGT

[0132] GmERE1-RNAi2R (SEQ ID NO.8): GTCAATTTGCAGGTATTTGGATCCCTGAATCTGGGAGGAGGTC

[0133] 2. Construction of the vector pFGC5941-GmERE1 for knocking down GmERE1 gene expression

[0134] Using the pMDTM18-T-GmERE1 plasmid obtained in Example 1 as a template, PCR amplification was performed to obtain the target fragment with the same restriction site as the vector. After sequencing was successful, the fragment was ligated into the vector pFGC5941 in both directions.

[0135] Example 3

[0136] Transformation and detection of GmERE1 transgenic complex plants:

[0137] I. Preparation of Agrobacterium competent cells by CaCl2 method

[0138] 1. Pick a single colony of LBA4404 and incubate it overnight on a 10m LYEP medium (containing 25mg / LRif) at 28℃ and 220rpm in a constant temperature shaker.

[0139] 2. In a clean bench, use a pipette to draw 800 μL of the bacterial culture for propagation, and then inoculate it a second time to activate it to OD. 600 It is approximately 0.6.

[0140] 3. Take an appropriate amount of bacterial culture into an EP tube and incubate on ice for 30 minutes, then centrifuge at 4000×g for 5 minutes at low temperature, and discard the waste liquid in the EP tube.

[0141] 4. Pre-cool the sterilized 0.05M CaCl2 solution at low temperature, use a pipette to take an appropriate amount of CaCl2 solution and repeatedly aspirate to precipitate, place the EP tube on ice for 25 minutes, centrifuge at 4000×g for 5 minutes, and discard the waste liquid in the EP tube.

[0142] 5. In a clean bench, take an appropriate amount of CaCl2 solution and repeatedly aspirate to precipitate the bacterial cells. Then, dispense the competent cells and store them at -80℃.

[0143] II. Freeze-thaw transformation of Agrobacterium

[0144] 1. Using a pipette, take 3 μL of the recombinant plasmids prepared in Examples 1 and 2 and transfer them into 100 μL of LBA4404 competent cells, then mix gently.

[0145] 2. Place the mixture on ice for 5 minutes, in liquid nitrogen for 5 minutes, incubate in a 37°C water bath for 5 minutes, and then immediately transfer it to an ice bath for 3 minutes.

[0146] 3. In a clean bench, use a pipette to take 800 μL of YEP culture medium and revive it for 4 hours at 28°C and 150 rpm in a constant temperature shaker.

[0147] 4. Centrifuge at 5,000×g for 2 min at room temperature, resuspend the bacterial cells with an appropriate amount of supernatant, take 200 μL of the revived bacterial solution and spread it evenly on the screening plate medium, and incubate upside down at 28℃ for 3 days.

[0148] III. Identification of Agrobacterium transformants

[0149] Single colonies were picked and placed in YEP liquid medium (Rif, Kan), plasmids were extracted, and enzyme digestion was performed for identification.

[0150] IV. Transformation of GmERE1 transgenic complex plants

[0151] Using the soybean cultivar "Dongnong 50" as the experimental material, "Dongnong 50" was planted in vermiculite and cultured in a light-temperature incubator (25℃, 16h light / 8h darkness) for 3 days. Then, K599 containing recombinant plasmid was activated, and fresh colonies from agar plates were used to create wounds on the hypocotyls of soybeans using a syringe for infection. After 21 days, newly germinated soybean hairy roots were examined, and positive soybean transgenic complex plants were obtained.

[0152] V. Detection of GmERE1 transgenic complex plants

[0153] 1. GFP fluorescence detection of hairy roots of GmERE1-overexpressing transgenic soybean

[0154] Irradiate hairy roots with GFP excitation light at a wavelength of 485 nm.

[0155] 2. Western blot analysis of hairy roots of GmERE1-overexpressing transgenic soybean

[0156] GmERE1-overexpressing transgenic soybean hairy roots that tested positive for GFP fluorescence were subjected to Western blot detection of the GmERE1-flag fusion protein using an anti-flag antibody. 0.2 g of GFP-positive hairy roots were thoroughly ground at ultra-low temperature, and an appropriate amount of protein buffer (100 mM HEPES, pH 7.5; 5 mM EDTA; 5% glycerol; 10 mM DTT; 1 mM PMSF; 10 μg / mL protease inhibitor) was added. After complete dissolution, the mixture was centrifuged at 4°C, 13000 rpm for 20 min. The supernatant was collected, and an appropriate amount of 5× protein loading buffer was added. After mixing, protein denaturation was performed (boiling water bath for 10 min).

[0157] (1) Preparation of main solutions:

[0158] 1) Electrophoresis buffer: 25mM Tris, 0.25M glycine, 0.1% SDS;

[0159] 2) Transfer buffer: 25mM Tris, 0.25M glycine, 0.1% SDS, 20% methanol;

[0160] 3) TBS buffer: 100mM Tris-HCl pH=7.5, 150mM NaCl;

[0161] 4) TBST buffer: TBS buffer containing 0.05% Tween 20;

[0162] 5) Blocking solution: TBST buffer containing 5% skim milk powder.

[0163] (2) SDS-PAGE electrophoresis

[0164] Take 20 μL of the denatured protein sample and perform electrophoresis on a stacking gel (60V). After transferring the bromophenol blue indicator to the separating gel, adjust the electrophoresis voltage to 120V. Stop electrophoresis when the bromophenol blue indicator migrates to the bottom of the separating gel.

[0165] (3) Transfer membrane

[0166] 1) Prepare a PVDF membrane of the same size as the adhesive and immerse the PVDF membrane in methanol for 3-5 minutes.

[0167] 2) Immerse two sponge pads, two 3mm filter papers, a glass rod, and the clamps for membrane transfer into the transfer solution.

[0168] 3) Assemble the transfer sandwich: Place the sponge pad, filter paper, glue, PVDF membrane, filter paper, and sponge pad in sequence on the black side of the clip. After each layer is placed, gently roll out the air bubbles with a glass rod, and then close and lock the clip.

[0169] 4) With the electrode orientation correctly aligned, place the locked clamp into the transfer tank and transfer in an 80V ice bath for 1.5 hours.

[0170] (4) Protein immune response

[0171] 1) Transfer the PVDF membrane to the blocking solution and shake it at room temperature for 1 hour to seal it.

[0172] 2) Transfer the PVDF membrane to a TBST solution containing the primary antibody (Anti-flag, abmart; M20008, diluted 5000 times) and incubate at room temperature for 1 hour.

[0173] 3) Clean the PVDF membrane with 10 mL of TBST, shake at room temperature for 10 min, repeat once, then clean the PVDF membrane with TBS, shake at room temperature for 10 min.

[0174] 4) Transfer the PVDF membrane to a TBST solution containing secondary antibody (pepper root peroxidase HRP, Merck Millipore) and incubate at room temperature for 1 hour.

[0175] 5) Clean the PVDF membrane with 10 mL of TBST, shake at room temperature for 10 min, repeat once, then clean the PVDF membrane with TBS, shake at room temperature for 10 min.

[0176] 6) Use filter paper to absorb the liquid on the membrane.

[0177] (5) Chemiluminescence

[0178] 1) Take 500 μL of each of the ECL solutions A and B, mix them, and add them to the membrane to fully wet the membrane.

[0179] 2) The membrane was placed in a chemiluminescence imaging system (Tianneng 5500) for analysis.

[0180] 3. qRT-PCR detection of hairy roots in GmERE1 transgenic soybean

[0181] (1) PCR detection of GmERE1 transgenic soybean plants

[0182] 1) Extraction of soybean genomic DNA (SDS micro-method)

[0183] ① Take 0.1g of hairy roots and grind them rapidly in liquid nitrogen. Add 400μL of extraction solution (500mM NaCl; 50mM EDTA, pH=8.0; 10mM β-mercaptoethanol; 100mM Tris-HCl, pH=8.0) and mix thoroughly.

[0184] ② Add 80 μL of 10% SDS and mix well.

[0185] ③ Incubate in a 65℃ water bath for 10 minutes, invert to mix, and repeat 3 times.

[0186] ④ Add 100 μL of 5M KAC, vortex for 6 seconds, and place on crushed ice for an ice bath for 30 minutes.

[0187] ⑤ Vortex for 20 seconds, add an equal volume of chloroform / isoamyl alcohol (24:1), and centrifuge at 12000 rpm for 15 min.

[0188] ⑥ Take 500 μL of the supernatant solution, add 500 μL of chloroform / isoamyl alcohol (24:1), invert to mix and extract DNA, and centrifuge at 12000 rpm for 10 min.

[0189] ⑦ Take 500 μL of the upper layer solution, add 500 μL of isopropanol, invert 5-6 times, and let stand at -20℃ for 30 min.

[0190] ⑧ Centrifuge at 12000 rpm for 5 min, discard the supernatant, add 70% alcohol and blow twice, let stand and dry, add 20-50 μL of deionized water to reconstitute, and store at -20℃.

[0191] (2) RT-PCR detection of GmERE1 transgenic hairy roots knocked down

[0192] 1) Primer design

[0193] DNA was extracted from hairy roots using the SDS method, and RT-PCR was used to detect whether the transformed plants carried the exogenous gene bar.

[0194] 2) Perform PCR detection on the bar gene.

[0195] 3) Detect the PCR amplification products by agarose gel electrophoresis.

[0196] Experimental results: such as Figure 1-4 As shown.

[0197] Depend on Figure 1 It was found that when a suitable amount of fresh soybean hairy roots were taken, and the hairy roots of GmERE1-OE transgenic soybean were detected using a GFP excitation light source and Western blot method, the GFP detection showed that all hairy roots of GmERE1-OE transgenic soybean were fluorescent green. The Western blot results are as follows: Figure 2 As shown: the target protein band was detected in the hairy roots of GmERE1-OE transgenic soybean, while no protein band was detected in EV. The GmERE1-RNAi transgenic hairy roots were detected using a Bar protein rapid test strip. Figure 3 As shown, both GmERE1-RNAi and EV (pFGC5941) transgenic soybean hairy roots tested positive. qPCR results indicated that the transcription level of GmERE1 in the hairy roots of GmERE1-OE transgenic soybean was significantly higher than that in EV (**P<0.01), while the transcription level of GmERE1 in the hairy roots of GmERE1-RNAi transgenic soybean was significantly lower than that in EV (**P<0.01). Figure 4 The above results indicate that GmERE1 transgenic soybean hairy roots and transgenic complex plants have been obtained.

[0198] Example 4

[0199] Drought resistance analysis of GmERE1 transgenic soybean complex plants:

[0200] 1. Preparation of test materials

[0201] The EV, GmERE1-OE, and GmERE1-RNAi transgenic soybean complexes obtained in Example 3 were subjected to drought stress by withholding irrigation for 8 days. The drought-resistant phenotypes of the transgenic lines were observed and photographed. Then, the wilted plants were rehydrated to restore growth, and the drought-resistant phenotypes of the transgenic soybean complexes were recorded 2 days later.

[0202] 2. Measurement of pore size

[0203] Collect the second and third leaves of soybean plants, immediately cover the lower epidermis of the leaves with transparent tape, and observe the morphology of the stomata under a microscope. Use ViewPoint software to measure the stomatal diameter in the images.

[0204] 3. Determination of superoxide dismutase activity

[0205] The SOD activity of hairy roots was determined using the WST-8 method of Ukeda and the superoxide dismutase (SOD) kit from Suzhou Keming Biotechnology Co., Ltd.

[0206] Place 0.1g of sample in a 1.5mL EP tube, add 1mL of crude enzyme extract, and homogenize in a cryogenic grinder. Centrifuge at 14000rpm, 4℃ for 10min. Transfer the supernatant to a new 1.5mL EP tube as the test sample. According to the kit instructions, mix 100μL of reagent 3 with 4.9mL of distilled water, label this as solution A1; mix 50mL of reagent 1 with 250μL of reagent 2, label this as solution A2; dissolve 100μL of reagent 4 in 5mL of water, label this as solution A3. Add 50μL of the test sample (50μL of distilled water for the control group) to a 1mL cuvette. 样 The reaction system consisted of 50 μL A1 solution, 800 μL A2 solution, and 100 μL A3 solution, with a total volume of 1 mL. After mixing and standing for 30 min, the absorbance of the control tube and the test tube at 450 nm was measured using a spectrophotometer, and recorded as A. 对照 and A 测定 .

[0207] The protein concentration (Cpr) of the samples was determined according to the instructions of the BCA Protein Assay Kit (Beyotime). A 0.5 mg / mL protein standard solution was prepared, and five 20 μL calibration systems were set up with protein concentrations of 0, 0.05, 0.2, 0.4, and 0.5 mg / mL, respectively. Simultaneously, an appropriate amount of the test sample was diluted to 20 μL. 200 μL of BCA working solution was added to the sample tubes containing the standard protein solution and the test sample, and the mixture was incubated at 37°C for 25 min. The sample tubes were then removed, and the absorbance of the reaction solution in each sample tube was measured at wavelengths of 540-595 nm using an ultra-micro analyzer. A protein standard curve was calculated with the protein standard solution concentration (mg / mL) on the x-axis and the absorbance on the y-axis. The corresponding protein concentration of the test sample was calculated using the absorbance value and recorded as Cpr (mg / mL).

[0208] The activity of SOD in the sample was calculated using the following formula:

[0209] Inhibition percentage (P) = [(A 对照 -A测定 ) / A 对照 ]×100%

[0210] SOD activity (U / mgprot) = [P / (1-P)×V] 反总 ] / (V 样 ×Cpr)

[0211] 4. Determination of peroxidase activity

[0212] Following the method of Kochba et al., the peroxidase (POD) kit from Suzhou Keming Biotechnology Co., Ltd. was used to determine the POD activity of hairy roots.

[0213] Place 0.1g of sample in a 1.5mL EP tube, homogenize with 1mL of crude enzyme extract at low temperature, centrifuge at 14000rpm, 4℃ for 10min, and use the supernatant as the test sample. Take 26mL of reagent one, 15μL of reagent two, and 10μL of reagent three from the kit into 50mL centrifuge tubes, mix well, and use as working solutions. Add 50μL of the test sample (V) to a 1mL cuvette according to the kit instructions. 样 Mix with 950 μL of working solution (V) 反总 The absorbance values ​​A1 and A2 at 470 nm for 1 min and 2 min respectively were measured using a spectrophotometer, respectively, indicating that the reaction time was 1 min.

[0214] The protein concentration (Cpr) of the sample was determined according to the instructions of the BCA Protein Assay Kit (Beyotime).

[0215] The activity of POD in the sample is calculated using the following formula:

[0216] POD(U / mgprot)=[(A2-A1)×V 反总 / (V 样 [×Cpr)]÷0.01 / T

[0217] Experimental results: The results are as follows Figure 5-9 As shown:

[0218] Five days after treatment, the opposite true leaves and trifoliate compound leaves of the GmERE1-OE transgenic soybean complex plants showed mild wilting, while the opposite leaves of the EV transgenic soybean complex plants showed mild wilting; the GmERE1-RNAi transgenic soybean complex plants showed no change. Eight days after treatment, all leaves of the GmERE1-OE transgenic soybean complex plants wilted, and the shoot apex meristem was severely atrophied. The opposite leaves of the EV transgenic soybean complex plants were severely wilted, and the trifoliate compound leaves were moderately wilted and turned yellow. The opposite leaves of the GmERE1-RNAi transgenic soybean complex plants were moderately wilted, and the trifoliate compound leaves were mildly wilted. Two days after rehydration, the meristems of the GmERE1-OE transgenic soybean complex plants and wild-type plants died and did not recover growth, while the meristems of the GmERE1-RNAi transgenic soybean complex plants showed good leaf meristem growth and the plants recovered growth. Figure 5 The above experiments demonstrate that overexpression of GmERE1 can reduce the drought resistance of soybean plants.

[0219] like Figure 6 and Figure 7 As shown, under moderate and severe drought treatments, the aspect ratio of stomatal diameter in the GmERE1-OE transgenic soybean complex was significantly higher than that in the EV transgenic soybean complex (*P<0.05), while the stomatal closure degree of the GmERE1-RNAi transgenic plants was significantly lower than that of the EV transgenic soybean complex. These results indicate that overexpression of GmERE1 under drought stress can increase water loss by inhibiting stomatal closure, thereby causing severe leaf wilting and reducing the drought resistance of the transgenic soybean complex.

[0220] like Figure 8 As shown: There was no significant difference in SOD activity between GmERE1-OE, GmERE1-RNAi, and EV transgenic soybean complex plants in the WW group; the SOD activity of GmERE1-RNAi in groups D1 and D2 was significantly higher than that of EV transgenic soybean complex plants (*P<0.05), while the SOD activity of GmERE1-OE was significantly lower than that of EV transgenic soybean complex plants (*P<0.05); there was no significant difference in SOD activity between GmERE1-OE, GmERE1-RNAi, and EV transgenic soybean complex plants in the RW group. These results indicate that under drought stress, GmERE1-RNAi can enhance the drought tolerance of soybeans by increasing the activity of SOD enzymes in soybean plants. Figure 9As shown: In the WW group, there was no significant difference in POD activity among GmERE1-OE, GmERE1-RNAi, and EV transgenic soybean complex plants; in the D1 and D2 groups, the POD activity of GmERE1-RNAi was significantly higher than that of the EV transgenic soybean complex plants (**P<0.01), while the POD activity of GmERE1-OE was significantly lower than that of the EV transgenic soybean complex plants (**P<0.01); in the RW group, there was no significant difference in POD activity among GmERE1-OE, GmERE1-RNAi, and EV transgenic soybean complex plants. These results indicate that under drought stress, GmERE1-RNAi can enhance the drought tolerance of soybean by increasing the activity of POD enzyme in soybean plants.

[0221] Example 5

[0222] Salt tolerance analysis of GmERE1 transgenic soybean complex plants:

[0223] The EV, GmERE1-OE and GmERE1-RNAi transgenic soybean complexes obtained in Example 3 were irrigated with water containing 0 mM NaCl and 250 mM NaCl. After 24 hours, samples were taken to determine the activities of their antioxidant enzymes SOD and POD.

[0224] Experimental results: such as Figure 10 and 11 As shown: After treatment with 250 mM NaCl, the SOD activity of the hairy roots of GmERE1-OE transgenic plants was significantly lower than that of EV (*P<0.05), while the SOD activity of the hairy roots of GmERE1-RNAi transgenic plants was significantly higher than that of EV (*P<0.05). Figure 10 The POD activity of GmERE1-OE transgenic hairy roots was significantly lower than that of EV (**P<0.01), while the POD activity of GmERE1-RNAi transgenic hairy roots was significantly higher than that of EV (**P<0.01). Figure 11 This indicates that overexpression of GmERE1 can inhibit antioxidant enzyme activity, thereby reducing the tolerance of transgenic soybean hairy roots to NaCl.

[0225] Example 6

[0226] Analysis of the GmERE1 combined with the GmPOD1 promoter:

[0227] I. Transient Expression Analysis of Tobacco

[0228] To determine whether GmERE1 can specifically bind to the DRE element, p35S:REN-DRE:LUC and EV empty effect vectors, reporter vector p35S:REN-DRE:LUC and effect vector p35S:GmERE1 were co-transfected into tobacco leaves. The specific steps are as follows:

[0229] 1. Construct p35S:REN-DRE:LUC and EV empty effect vectors, reporter vector p35S:REN-DRE:LUC and effect vector p35S:GmERE1.

[0230] 2. Agrobacterium-mediated injection into tobacco leaves

[0231] (1) Tobacco cultivation: The seeds of Tobacco Benedict were evenly scattered in soil with a vermiculite-soil ratio of 1:1 and placed in a greenhouse incubator with a temperature of 25℃, 16h light / 8h darkness. Water was applied every two days. When the leaves had unfolded, leaves with good growth were selected for Agrobacterium injection.

[0232] (2) Prepare osmotic buffer (10mM MgCl2, 1mM MES, 150μM acetylsylcholine, pH=5.7).

[0233] (3) Agrobacterium-injected tobacco

[0234] 1) Take 50 μL of GV3101 glycerol bacteria transformed with the pGmERE1-pGreenII 0800 recombinant plasmid and place it in 50 mL of YEP liquid medium containing the corresponding antibiotic. Incubate overnight at 28°C with shaking at 220 rpm. Then, activate the bacteria again. When the OD600 value of the bacterial culture is 0.8, centrifuge at 8000 rpm for 10 min and collect the bacterial cells.

[0235] 2) Resuspend the Agrobacterium cells in 10 mL of osmotic buffer, centrifuge at 8000 rpm for 5 min, remove the supernatant, and repeat three times.

[0236] 3) Adjust the OD600 value of the tobacco infection solution to 0.6 with osmotic buffer and allow it to stand in the dark at room temperature for 2-3 hours for recovery.

[0237] 4) Mix the Agrobacterium infection solution containing GmERF5-pCAMBIA3301 and pCAMBIA3301 with the Agrobacterium infection solution containing pGmERE1-pGreenII 0800 at a ratio of 1:1, and inject the two mixed Agrobacterium infection solutions into tobacco leaves in good growth condition.

[0238] 5) Cover the injected tobacco plants with an opaque cardboard box to protect them from light and incubate them at room temperature for 3 days.

[0239] 6) Prepare 1mM luciferin in a small spray bottle, spray luciferin onto the entire tobacco leaf, and leave in the dark for 10 minutes.

[0240] 7) Analysis was performed using a chemiluminescence imaging system (Tianneng 5500).

[0241] (4) Detection of LUC activity

[0242] The activity of LUC in tobacco leaves was detected using a Dual Luciferase assay kit (Promega).

[0243] II. Chromatin Immunoprecipitation (ChIP-qPCR)

[0244] The relationship between the GmERE1 gene and the GmPOD1 gene was verified using chromatin immunoprecipitation (ChIP-qPCR).

[0245] 1. Preparation of test reagents

[0246] (1) MC buffer: 10 mM potassium phosphate buffer (pH = 7.0), 50 mM sodium chloride (NaCl), 0.1 M sucrose; 2) M1 buffer: 10 mM potassium phosphate buffer (pH = 7.0), 0.1 M sodium chloride (NaCl), 1 M hexylene glycol, 10 mM beta-mercaptoethanol; 3) M2 buffer: 10 mM potassium phosphate buffer (pH = 7.0), 1 mM magnesium chloride (MgCl2), 0.1 M sodium chloride (NaCl), 0.50% Tritium X-100, 10 mM beta-mercaptoethanol, 1 M hexylene glycol 4) M3 buffer: 10 mM potassium phosphate buffer (pH=7.0), 0.1 M sodium chloride (NaCl), 10 mM β-mercaptoethanol; 5) Ultrasonic buffer: 10 mM potassium phosphate buffer (pH=7.0), 0.1 M sodium chloride (NaCl), 10 mM ethylenediamine (EDTA, pH=0.8), 0.50% sodium dodecyl sarcosinate; 6) IP buffer: 50 mM hydroxyethylpiperazine ethanethioic acid (Hepes, pH=7.5), 150 mM potassium chloride (KCl), 50 mM magnesium chloride (MgCl2), 10 μM zinc sulfate (ZnSO4), 1% Tritium X-100; 0.05% sodium dodecyl sulfate (SDS); 7) Elution buffer: 50mM hydroxyethylpiperazine ethanethioic acid (Hepes, pH=7.5), 10% sodium dodecyl sulfate (SDS), 1M Tris-HCl (pH=8.0), 0.5M ethylenediamine (EDTA).

[0247] 2. Specific steps

[0248] (1) Place 1g of fresh transgenic soybean hairy roots overexpressing GmERE1 and empty vector into MC buffer containing 1% formaldehyde, place in a vacuum chamber, and vacuum permeate the sample. After fixing the sample for 30 min, add 0.41662g of glycine powder (final concentration of glycine powder is 0.15M) to terminate the fixation reaction. Remove the MC buffer, add fresh pre-cooled MC buffer, and wash the sample 3 times. Quickly place the sample on filter paper to absorb the MC buffer on its surface, transfer the sample into a 50mL centrifuge tube, flash freeze in liquid nitrogen, and store at -80℃ for use in nucleoprotein-DNA extraction.

[0249] (2) Prepare M1 buffer containing PMSF (final PMSF concentration 1 mM) and pre-cool on ice. Grind the sample in liquid nitrogen to a fine powder, then resuspend the sample thoroughly in the prepared M1 buffer. Transfer the resuspended sample to a 2 mL EP tube and centrifuge at 12,000 rpm for 3 min at 4 °C. Prepare M2 buffer and add PMSF (to a final PMSF concentration of 1 mM), pre-cool on ice, and thoroughly resuspend the precipitate in M2 buffer. Shake at 4 °C for 20 min, then centrifuge at 12,000 rpm for 3 min at 4 °C; discard the supernatant. Repeat several times until the supernatant is nearly transparent and colorless. Prepare M3 buffer containing PMSF (final PMSF concentration 1 mM), pre-cool on ice, and thoroughly resuspend the precipitate in M3 buffer. Centrifuge at 12,000 rpm for 3 min at 4 °C. Discard the supernatant and repeat the steps once.

[0250] (3) Prepare an ultrasonic buffer (containing 1 mM PMSF and 1 times the amount of protease inhibitor), and resuspend the sample precipitate in 500 μL of ultrasonic buffer. Place the resuspended sample on ice and sonicate for 10 min (ultrasonic program: sonicate for 15 s, pause for 15 s, frequency 20%). Centrifuge the sonicated sample at 4°C, 12000 rpm for 5 min, and collect the supernatant into a new EP tube. Resuspend the precipitate again in 250 μL of ultrasonic buffer until the precipitate and ultrasonic buffer are homogenized, then centrifuge at 4°C, 12000 rpm for 5 min, and collect the supernatant into a new EP tube, for a total of 750 μL of sample. Extract 75 μL of the obtained sample as input sample and freeze at -20°C for DNA analysis.

[0251] (4) Prepare IP buffer (containing 1 mM PMSF and 1-fold protease inhibitor) and pre-cool on ice. Add an equal volume of IP buffer and 30 μL of protein agarose beads (Protein A / Gagarose) containing the corresponding antibody to the sonicated sample, and incubate at 4°C for 8-16 h by vortexing to ensure sufficient binding of the sample with Protein A / Gagarose. Centrifuge at 2500 rpm for 2 min at 4°C, remove the supernatant, and retain Protein A / Gagarose. Resuspend Protein A / Gagarose in 1 mL of IP buffer (containing 1-fold protease inhibitor), and incubate at 4°C for 5 min by vortexing. Repeat 3 times. Centrifuge at 2500 rpm for 2 min at 4°C, remove the supernatant, add 200 μL of elution buffer, vortex vigorously, and incubate at 65°C for 15 min. Then centrifuge at 12000 rpm for 1 min at 25°C, and transfer the supernatant to a new 1.5 mL EP tube. Add 150 μL of elution buffer to the precipitate after centrifugation, and repeat twice. Elute the sample (500 μL) by centrifugation at 12000 rpm for 2 min at 25 °C. Take 25 μL of the supernatant for Western blot analysis, and use the remainder for DNA analysis.

[0252] (5) Reverse cross-linking reaction: Add 5M NaCl to the eluted sample (to a final NaCl concentration of 0.3M), and incubate overnight in a 65℃ water bath. Add 1-2 μL of RNase A, and incubate for 30 min in a 37℃ water bath. Add proteinase K to a final concentration of 0.5 mg / mL; incubate for 1 h in a 45℃ water bath. Add an equal volume of chloroform:isoamyl alcohol (24:1), mix well, and centrifuge at 12000 rpm for 10 min. Transfer the supernatant to a new 1.5 mL EP tube and add 1 / 10 volume of 3M sodium acetate and 2.5 volumes of anhydrous ethanol, and incubate overnight at -20℃. Centrifuge at 14000 rpm for 20 min at 4℃. Wash the precipitate twice with 70% ethanol, dry it, and add 50 μL of ddH2O to dissolve the DNA.

[0253] 3. qRT-PCR detection of target genes

[0254] (1) Primer design

[0255] Primers were designed at the P1 position (containing one DRE element) and the P2 position (not containing DRE or ERE elements) of GmPOD1, and the primer sequences are shown in Table 1.

[0256] Table 1. GmPOD1 ChIP-qPCR Primer Sequences

[0257]

[0258] (2) qRT-PCR detection

[0259] For specific operating procedures, please refer to Example 1.

[0260] The results are as follows Figure 12-13 As shown:

[0261] Compared with tobacco leaves co-transfected with reporter vector p35S:REN-DRE:LUC and EV empty effect vector, the chemiluminescent signal detected in tobacco parts co-transfected with reporter vector p35S:REN-DRE:LUC and effect vector p35S:GmERE1 was significantly suppressed, indicating that GmERE1 can bind to the DRE element and inhibit the expression of downstream genes. The LUC / REN relative activity assay further showed that GmERE1 can significantly inhibit the expression of downstream genes by binding to the DRE element (**P<0.01). Figure 12 ).

[0262] In GmERE1-OE transgenic hairy roots, the enrichment levels of GmERE1 protein and the P1 region of the GmPOD1 promoter were significantly higher than those in EV hairy roots (**P<0.01), while no difference in enrichment was detected in the P2 region of the DRE-free promoter. This indicates that GmERE1 can directly bind to the DRE-containing region of the GmPOD1 promoter. Figure 13 ).

[0263] Example 7

[0264] Analysis of GmPOD1 expression levels in GmERE1 transgenic hairy roots under drought and salt stress:

[0265] For specific instructions, please refer to Examples 1-5.

[0266] The results are as follows Figure 14 As shown:

[0267] The relative expression level of GmPOD1 in the hairy roots of GmERE1-OE transgenic plants treated with drought and 250 mM NaCl was significantly lower than that in EV (**P<0.01), while the relative expression level of GmPOD1 in the hairy roots of GmERE1-RNAi transgenic plants was significantly higher than that in EV (**P<0.01). Figure 14 These results indicate that GmERE1 may affect POD synthesis by directly inhibiting the expression of GmPOD1, thereby reducing the drought and salt tolerance of plants.

[0268] Example 8

[0269] Transient expression analysis of tobacco: GmERF5 inhibits GmERE1 expression.

[0270] The following uses GmERF5, Phytozome accession no. Glyma.14G050100.

[0271] 1. Cloning of the GmERE1 promoter and construction of vectors

[0272] Primers were designed based on the 2000bp upstream sequence of the GmERE1 transcription start site to amplify the GmERE1 promoter. The primer sequences are as follows:

[0273] GmERE1aP-LUC F (SEQ ID NO. 13): CGGGCCCCCCCTCGAGGTCGACCGCTTTTTAACCTTTCCCTG;

[0274] GmERE1aP-LUC R (SEQ ID NO. 14): CCGCTCTAGAACTAGTTTTGTTGCTGTT TTGGCTG.

[0275] The GmERE1 promoter region was ligated to the pGreenII 0800-LUC vector, with Sal1 and BamH1 restriction sites; the recombinant plasmid pGmERE1-pGreenII 0800 was transformed into Agrobacterium GV3101. The specific procedure is described in Example 3.

[0276] 2. Agrobacterium-mediated injection into tobacco leaves

[0277] (1) Tobacco cultivation: The seeds of Tobacco Benedict were evenly scattered in soil with a vermiculite-soil ratio of 1:1 and placed in a greenhouse incubator with a temperature of 25℃, 16h light / 8h darkness. Water was applied every two days. When the leaves had unfolded, leaves with good growth were selected for Agrobacterium injection.

[0278] (2) Prepare osmotic buffer (10mM MgCl2, 1mM MES, 150μM acetylsylcholine, pH=5.7).

[0279] (3) Agrobacterium-injected tobacco

[0280] 1) Take 50 μL of GV3101 glycerol bacteria transformed with the pGmERE1-pGreenII 0800 recombinant plasmid and place it in 50 mL of YEP liquid medium containing the corresponding antibiotic. Incubate overnight at 28°C with shaking at 220 rpm. Then, perform a second activation and wait for the OD of the bacterial culture to reach the specified value. 600 The value was 0.8, and the cells were collected by centrifugation at 8000 rpm for 10 min.

[0281] 2) Resuspend the Agrobacterium cells in 10 mL of osmotic buffer, centrifuge at 8000 rpm for 5 min, remove the supernatant, and repeat three times.

[0282] 3) Use osmotic buffer to dilute the OD of the tobacco staining solution. 600 Adjust the value to 0.6 and allow it to recover in the dark at room temperature for 2-3 hours.

[0283] 4) Mix the Agrobacterium infection solution containing GmERF5-pCAMBIA3301 and pCAMBIA3301 with the Agrobacterium infection solution containing pGmERE1-pGreenII 0800 at a ratio of 1:1, and inject the two mixed Agrobacterium infection solutions into tobacco leaves in good growth condition.

[0284] 5) Cover the injected tobacco plants with an opaque cardboard box to protect them from light and incubate them at room temperature for 3 days.

[0285] 6) Prepare 1mM luciferin in a small spray bottle, spray luciferin onto the entire tobacco leaf, and leave in the dark for 10 minutes.

[0286] 7) Analysis was performed using a chemiluminescence imaging system (Tianneng 5500).

[0287] (4) Detection of LUC activity

[0288] The activity of LUC in tobacco leaves was detected using a Dual Luciferase assay kit (Promega).

[0289] The results are as follows Figure 15 and 16 As shown:

[0290] Compared with the reporter vector p35S:REN-GCC:LUC and the empty EV effect vector co-transfected tobacco leaves, the chemiluminescent signal detected in tobacco parts co-transfected with the reporter vector p35S:REN-GCC:LUC and the effect vector p35S:GmERF5-myc was significantly suppressed, indicating that GmERF5 can bind to the GCC-box and inhibit the expression of downstream genes. Figure 15 The results of the LUC / REN relative activity assay further indicate that GmERF5 can significantly inhibit the expression of downstream genes by binding to the GCC-box element (**P<0.01). Figure 15 (b)

[0291] The chemiluminescent signal detected in the tobacco co-transfected sites of the p35S:GmERF5-myc / pGmERE1:LUC group was significantly suppressed compared with that of the p35S / pGmERE1:LUC group. Figure 16 In a), and the relative activity of LUC / REN enzyme in the p35S:GmERF5-myc / pGmERE1:LUC combination was significantly lower than that in the p35S / pGmERE1:LUC group (**P<0.01). Figure 16 (b) The above results further demonstrate that GmERF5 directly inhibits the expression of GmERE1.

[0292] As demonstrated by the above embodiments, overexpression of GmERE1 in this invention can inhibit antioxidant enzyme activity, thereby reducing the drought resistance and salt tolerance of transgenic complex plants. The GmERF5 gene can specifically bind to the GCC-box element on the GmERE1 promoter, directly regulating and inhibiting GmERE1 expression. The combination of GmERF5 and GmERE1 can promote GmPOD1 expression, increasing peroxidase activity in soybean plants and thus improving drought resistance and salt tolerance. The soybean GmERF5 and GmERE1 of this invention provide a novel regulatory gene resource for improving soybean drought resistance and salt tolerance.

[0293] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of the GmERE1 gene in altering the drought resistance and / or salt tolerance of soybeans, characterized in that, Knocking down the expression of the GmERE1 gene in soybean can improve the drought resistance and / or salt tolerance of soybean. The nucleotide sequence of the GmERE1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the GmERE1 gene is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The method for knocking down GmERE1 gene expression includes the following steps: The GmERE1 gene was cloned into the pFGC5941 vector to obtain the recombinant vector; The obtained recombinant vector was transformed into Agrobacterium rhizogenes, and the transformed Agrobacterium rhizogenes was used to infect seeds to obtain transgenic complex plants, thereby achieving the knockdown expression of the GmERE1 gene.

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