Application of GmERF5 gene in drought and salt stress resistance

Abstract

The application provides application of a GmERF5 gene in drought resistance and salt stress resistance, and belongs to the technical field of genetic engineering.The GmERF5 gene is cloned into a pCAMBIA3301 vector to obtain a recombinant vector; the obtained recombinant vector is transformed into Agrobacterium rhizogenes, and a transgenic complex plant is obtained by using the obtained Agrobacterium rhizogenes to infect seeds, so that overexpression of the GmERF5 gene is realized.The overexpression of the GmERF5 gene can improve the antioxidant enzyme activity of the transgenic soybean plant, and the drought and salt stress resistance of the soybean is enhanced.The soybean GmERF5 of the application provides a new regulation gene resource for improving the drought resistance and salt tolerance of soybean, can be used for cultivation and improvement of stress-resistant soybean materials, lays a theoretical foundation for drought resistance and salt molecular mechanism, and simultaneously provides a theoretical basis and gene resource for soybean drought resistance and salt molecular breeding.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to the application of a GmERF5 gene in drought resistance and salt tolerance. Background Technology

[0002] Soybean (Glycine max L.) is an important food and economic crop. Soybeans have underdeveloped root systems 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, easily leading to leaf wilting, yellowing, curling, and stunted growth, thus affecting yield and quality, and in severe cases, causing plant death. Biological and abiotic stresses, including pests and diseases, low temperatures, drought, and soil salinization, pose serious threats to soybean production. Therefore, enhancing stress resistance and cultivating germplasm resources with better agronomical traits is urgently needed. While traditional breeding methods have achieved significant results, research is limited by long breeding cycles, the complexity of stress resistance mechanisms, and the lack of superior germplasm resources. Combining modern molecular biology techniques with traditional breeding methods to study plant stress resistance mechanisms has become an inevitable choice for many breeders.

[0003] The abiotic stress network in plants is extremely complex, containing numerous gene-level transcriptional regulations. Plants regulate their physiological development by transcriptionally regulating relevant stress genes. Environmental stimuli such as ion stress, pathogens, water, and temperature affect plant signal transduction. To avoid damage from abiotic stress, plants have developed a set of self-regulatory mechanisms to protect themselves from adverse environmental pressures such as low temperature, drought, and high salinity. AP2 / ERF transcription factors play important roles in plant stress responses, especially in drought and salt stress responses. ERF-TFs are widely involved in the abiotic stress response processes of many species, including drought and salt. GmERF3, TSRF1, OsERF48, and TaERF3 have been identified as positive regulators of drought resistance in soybean, rice, and wheat, respectively. 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 rates. Several ERFs in wheat have been identified, and their regulation may be used for the development of improved varieties. Chinese patent CN114540367A (Application of soybean GmPRR3b gene in regulating soybean drought resistance, 2022.2.18) found that the soybean GmPRR3b gene can negatively regulate soybean drought resistance. Chinese patent CN114085844A (Application of soybean salt tolerance gene GmERD15B, 2020.7.13) indicates that overexpression of GmERD15B may enhance soybean salt tolerance by increasing the expression levels of genes known to be related to salt stress (such as ABA signaling, dehydration response, and ion transport).

[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 an application of the GmERF5 gene in drought resistance and salt tolerance.

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

[0007] This invention provides an application of the GmERF5 gene in altering plant drought resistance and / or salt tolerance. Overexpression of the GmERF5 gene in plants enhances their drought resistance and / or salt tolerance; knockdown of the GmERF5 gene in plants reduces their drought resistance and / or salt tolerance.

[0008] Preferably, the nucleotide sequence of the GmERF5 gene is shown in SEQ ID NO.1.

[0009] Preferably, the amino acid sequence of the protein encoded by the GmERF5 gene is shown in SEQ ID NO.2.

[0010] Preferably, the method for overexpressing the GmERF5 gene includes the following steps:

[0011] The GmERF5 gene was cloned into the pCAMBIA3301 vector to obtain the recombinant vector;

[0012] 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 GmERF5 gene.

[0013] Preferably, the method for knocking down GmERF5 gene expression includes the following steps:

[0014] The GmERF5 gene was cloned into the pFGC5941 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 the knockdown expression of the GmERF5 gene.

[0016] The present invention also provides a recombinant vector for overexpressing the GmERF5 gene, the recombinant vector comprising an initial expression vector and the GmERF5 gene.

[0017] Preferably, the initial expression vector is the pCAMBIA3301 vector.

[0018] The present invention also provides a recombinant vector for knocking down the expression of the GmERF5 gene, the recombinant vector comprising the pFGC5941 vector and the GmERF5 gene.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention demonstrates that overexpression of the GmERF5 gene can enhance the antioxidant enzyme activity of transgenic soybean plants, thereby increasing soybean's tolerance to drought and salt stress. The soybean GmERF5 gene of this invention provides a novel regulatory gene resource for improving soybean drought and salt tolerance, which can be used for the breeding and improvement of stress-resistant soybean materials, laying a theoretical foundation for the molecular mechanisms of drought and salt tolerance, and also providing theoretical basis and gene resources for molecular breeding of soybeans for drought and salt tolerance. Attached Figure Description

[0021] Figure 1 This represents the relative expression level of GmERF5 under 10% PEG6000 treatment;

[0022] Figure 2 This represents the relative expression level of GmERF5 under 250 mM NaCl treatment;

[0023] Figure 3 This is an agarose gel electrophoresis result of double digestion with GmERF5 and vector pCAMBIA3301;

[0024] Figure 4 This is a graph showing the results of enzyme digestion identification and sequencing analysis of GmERF5 and vector pFGC5941;

[0025] Figure 5 This is a graph showing the results of SDS-PAGE assay on GmERF5-OE transgenic plants.

[0026] Figure 6 This is a Western blot result of GmERF5-OE transgenic plants;

[0027] Figure 7 This is a graph showing the results of SDS-PAGE assay on GmERF5-RNAi transgenic plants.

[0028] Figure 8 This is a Western blot result of GmERF5-RNAi transgenic plants;

[0029] Figure 9 This represents the expression level of GmERF5 in GmERF5-OE transgenic plants and GmERF5-RNAi transgenic plants.

[0030] Figure 10 This is a schematic diagram showing the growth status of GmERF5-OE and GmERF5-RNAi transgenic plants under drought treatment.

[0031] Figure 11 This is a schematic diagram of stomatal morphology in GmERF5-OE and GmERF5-RNAi transgenic plants under drought treatment.

[0032] Figure 12 The stomatal cross-sectional / vertical ratio of GmERF5-OE and GmERF5-RNAi transgenic plants under drought treatment;

[0033] Figure 13 The SOD activity of GmERF5-OE and GmERF5-RNAi transgenic plants under drought treatment;

[0034] Figure 14 The POD activity of GmERF5-OE and GmERF5-RNAi transgenic plants under drought treatment;

[0035] Figure 15This is a schematic diagram showing the growth of the radicle of GmERF5-OE and GmERF5-RNAi transgenic soybean seeds under treatment with 0, 150, and 250 mM NaCl.

[0036] Figure 16 The length of the radicle in GmERF5-OE and GmERF5-RNAi transgenic soybean seeds after treatment with 0, 150, and 250 mM NaCl;

[0037] Figure 17 The SOD activity of GmERF5-OE and GmERF5-RNAi transgenic plants under 250mM NaCl treatment;

[0038] Figure 18 The POD activity of GmERF5-OE and GmERF5-RNAi transgenic plants under 250mM NaCl treatment;

[0039] Figure 19 This represents the expression level of GmPOD1 in GmERF5-OE and GmERF5-RNAi transgenic plants under drought and salt stress. Detailed Implementation

[0040] This invention provides an application of the GmERF5 gene in altering plant drought resistance and / or salt tolerance. Overexpression of the GmERF5 gene in plants enhances their drought resistance and / or salt tolerance; knockdown of the GmERF5 gene in plants reduces their drought resistance and / or salt tolerance.

[0041] In this invention, the nucleotide sequence of the GmERF5 gene is as shown in SEQ ID NO.1, specifically:

[0042] In this invention, the amino acid sequence of the protein encoded by the GmERF5 gene is shown in SEQ ID NO.2, specifically as follows:

[0043] MRRGRATAAVVDPTAEQAKETRFRGVRKRPWGRFAAEIRDPWKKQRVWLGTFDSAEDAARAYDKAARSFRGPKAKTNFPSFPGPTDHHSSQQIPPLYQAHGLSTKF EPAQVNRPTTSGMSSTVESFSGPRVPPSSSSRKPLVVVNPIIPLDDDDDDCHSDCDSSSSVVDDQDCVLTSSFRQPLPFDLNLPPPDAAYDDDDDDVPATALCL.

[0044] Preferably, the method for overexpressing the GmERF5 gene includes the following steps:

[0045] The GmERF5 gene was cloned into the pCAMBIA3301 vector to obtain the recombinant vector;

[0046] 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 GmERF5 gene.

[0047] 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.

[0048] Preferably, the method for knocking down GmERF5 gene expression includes the following steps:

[0049] The GmERF5 gene was cloned into the pFGC5941 vector to obtain the recombinant vector;

[0050] 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 GmERF5 gene.

[0051] 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.

[0052] This invention also provides a recombinant vector overexpressing the GmERF5 gene, wherein the recombinant vector comprises an initial expression vector and the GmERF5 gene. In this invention, the initial expression vector is the pCAMBIA3301 vector (http: / / www.biofeng.com / ). The construction method of the recombinant vector involves ligating the GmERF5 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 vector plasmid to obtain the recombinant vector overexpressing the GmERF5 gene.

[0053] This invention also provides a recombinant vector for knocking down the expression of the GmERF5 gene, the recombinant vector comprising the pFGC5941 vector (http: / / www.biofeng.com / ) and the GmERF5 gene. In this invention, the recombinant vector is constructed by using the GmERF5 sequence as a template for homology comparison to find its homologous gene in soybean, designing specific primers to amplify the dissimilar portion of GmERF5 and 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.

[0054] 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.

[0055] Example 1

[0056] Expression of the GmERF5 gene under drought and salt stress:

[0057] 1. Cultivation and treatment of experimental materials

[0058] Plump, disease-free soybean cultivar “Dongnong 50” was selected and planted in round pots containing vermiculite-mixed humus. The pots were then placed in a light-temperature incubator at 25℃ with 16h light / 8h darkness. When the soybean seedlings reached the V3 stage, they were treated with 10% PEG6000 and 250mM NaCl according to the method of Li et al. (Li W, Wang T, Zhang Y, Li Y. Overexpression of soybean miR172c conferstolerance to water deficit and salt stress, but increases ABA sensitivity in transgenic Arabidopsis thaliana[J]. Journal of Experimental Botany, 2016, 67(1): 175-194). At different time points, the trifoliate leaves of the soybean seedlings were taken, quick-frozen in liquid nitrogen, and then stored in a -80℃ freezer for RNA extraction.

[0059] 2. Synthesis of total RNA and cDNA extracted from soybeans using the Trizol Reagent method

[0060] (1) RNA extraction

[0061] 1) Place an appropriate amount of soybean leaves required for the experiment into the grinding tube provided by the cryogenic grinder (Hangzhou Suizeng Biotechnology Co., Ltd.), add 1 mL of Trizol reagent, and grind in the cryogenic grinder for 1 min.

[0062] 2) Add chloroform, shake for 15 seconds, place on ice for 3 minutes, centrifuge at 4°C and 12,000 rpm for 10 minutes.

[0063] 3) Take 500 μL of the upper layer solution, mix it thoroughly with 500 μL of isopropanol, incubate on ice for 10 min, and then centrifuge at 4℃ and 12000 rpm for 10 min.

[0064] 4) Discard the supernatant, gently blow up the RNA precipitate at the bottom of the EP tube with 75% ethanol (RNase removed), and centrifuge at 7500 rpm for 5 min at 4°C. Repeat once.

[0065] 5) Remove the supernatant, and after the RNA precipitate dries, add 20-40 μL of DEPC water. After the precipitate is completely dissolved, store it at -80℃ for use in cDNA synthesis.

[0066] (2) cDNA synthesis

[0067] 1) Take 4 μL of RNA into a siliconized PCR tube and denature it at 65°C for 5 min.

[0068] 2) Add 2 μL of 4×DN Master Mix and 2 μL of water in sequence, and react at 37℃ for 5 min.

[0069] 3) Add 2 μL of 5×RT Master MixⅡ, and store the cDNA at 37℃ for 15 min, 50℃ for 5 min, 98℃ for 5 min, and -20℃.

[0070] 3. Quantitative real-time PCR (qRT-PCR) detection

[0071] (1) Design of qRT-PCR primers

[0072] 1) Design quantitative fluorescence primers based on the CDS sequence of GmERF5:

[0073] F(SEQ ID NO.3):TGAGCAGCACCGTGGAGTC

[0074] R(SEQ ID NO.4):GCGGCGGGAGGTTTAGAT

[0075] 2) Design quantitative real-time primers based on the CDS sequence of the internal reference gene GmEF1β:

[0076] F(SEQ ID NO.5):CCACTGCTGAAGAAGATGATGATG

[0077] R(SEQ ID NO.6):AAGGACAGAAGACTTGCCACTC

[0078] (2) Specific steps of qRT-PCR

[0079] The TOYOBOSYBR GREEN (QPS-201) kit was used in the experiment. The reaction system is as follows:

[0080] ddH2O: 5.6 μL

[0081] Primer F: 1.2μL

[0082] Primer R: 1.2μL

[0083] 2×SYBR Green: 10μL

[0084] cDNA: 2μL

[0085] Total volume: 20μL

[0086] The amplification program was: 95℃ for 3 min; 95℃ for 30 sec, 60℃ for 30 sec, for 30 cycles. Results were presented using 2... -△△Ct Quantitative calculations are performed using the method. △△Ct = (CtGmERF5 - CtGmEF1β) before treatment - (CtGmERF5 - CtGmEF1β) after treatment.

[0087] The results are as follows Figure 1 and Figure 2 As shown:

[0088] The relative expression level of GmERF5 was significantly higher at 9-72 h after treatment with 10% PEG6000 than at 0 h (*P<0.05), and reached a highly significant level at 12-36 h (**P<0.01), with the highest value at 12 h (7.26 times that of the control group). Figure 1 The above results indicate that GmERF5 expression is induced by drought stress.

[0089] The relative expression level of GmERF5 was significantly higher at 6-72 h after treatment with 250 mM NaCl than at 0 h (*P<0.05), and reached a highly significant level at 6-36 h (**P<0.01), with the highest value at 12 h (9.87 times that of the control group). Figure 2 The above results indicate that GmERF5 expression is induced by salt stress.

[0090] Example 2

[0091] Ligation and transformation of plant expression vectors:

[0092] 1. Construction of plant overexpression vector pCAMBIA3301-GmERF5

[0093] (1) Primer design

[0094] The overexpression recombinant vector pCAMBIA3301-GmERF5 was constructed. Using the cDNA of soybean cultivar "Dongnong 50" as a template, specific primers were designed, and RT-PCR amplification was performed to obtain the full-length GmERF5 fragment. This fragment was then ligated into the pMDTM18-T vector. The recombinant plasmid and pCAMBIA3301 plasmid, which had been identified by sequencing, were digested with enzymes at Nco1 and Pml1. The digested product containing the GmERF5 fragment was then ligated into pCAMBIA3301 to obtain the pCAMBIA3301-GmERF5 recombinant vector.

[0095] The primer sequences are as follows:

[0096] F (SEQ ID NO.7): GCCATGGAGATGCGCCGAGGGAGAG

[0097] R(SEQ ID NO.8):GCACGTGGAGGCACAGCGCGGTGGC

[0098] (2) Linkage reaction of T-carrier

[0099] The target fragment was ligated into the pMDTM18-T vector, and the ligation reaction system was as follows:

[0100] Solution I: 4μL

[0101] pMDTM18-T: 1μL

[0102] Gel recovery product: 5μL

[0103] Total volume: 10μL

[0104] After thorough mixing, react at 16°C for 4 hours.

[0105] (3) Conversion of the linker products

[0106] 1) Thaw E. coli DH5α competent cells on ice.

[0107] 2) Add the product obtained in step (2) to E. coli DH5α competent cells and incubate on ice for 30 min.

[0108] 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.

[0109] 4) Add 700 μL of LB liquid, place in a shaker, and incubate at 37°C and 220 rpm for 60 min.

[0110] 5) Take 200 μL of the bacterial suspension and spread it evenly on a screening plate. Incubate at 37°C upside down overnight.

[0111] (4) PCR identification of transformed clones

[0112] 1) Select plump single spots and culture them in liquid screening medium at 37℃ and 220rpm for 4-6 hours.

[0113] 2) Bacterial culture PCR identification was performed using Mix enzymes. The PCR system is as follows:

[0114] 2×Taq Master Mix enzyme: 12.5μL

[0115] Bacterial solution: 1 μL

[0116] Primer F (10 μM): 1 μL

[0117] Primer R (10 μM): 1 μL

[0118] ddH2O: 9.5 μL

[0119] Total volume: 25μL

[0120] 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℃.

[0121] 4) Detect the PCR amplification products by electrophoresis, select the bacterial solutions that are identified as positive, and use them for plasmid extraction.

[0122] (5) Extraction of recombinant plasmids

[0123] 1) Take 2 mL of bacterial culture and centrifuge at 12000 rpm for 1 min.

[0124] 2) Add 250 μL of SI liquid to resuspend the bacterial pellet.

[0125] 3) Add 250 μL of SⅡ liquid and shake well several times.

[0126] 4) Add 350 μL of SⅢ liquid, shake well, and centrifuge at 12000 rpm for 10 min.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 8) Place the preparation tube back into the EP tube and centrifuge for 1 min.

[0131] 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℃.

[0132] (6) Double enzyme digestion identification of recombinant plasmids

[0133] The double enzyme digestion system is as follows:

[0134] Recombinant plasmid: 16 μL

[0135] 10×Buffer: 2μL

[0136] Endonuclease I: 1 μL

[0137] Endonuclease II: 1 μL

[0138] Total volume: 20μL

[0139] React at 37℃ for 30 min, then perform 1% agarose gel electrophoresis for detection.

[0140] (7) Sequencing

[0141] 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.

[0142] (8) Construction of the carrier

[0143] 1) Perform double digestion of the recombinant plasmid that has been sequenced without error and the pCAMBIA3301 plasmid, respectively, and refer to step (6) for the system and reaction conditions.

[0144] 2) Recover the target fragment and the digested vector fragment according to the instructions of the OMEGA gel recovery kit.

[0145] 3) The ligation of the target gene DNA fragment with the vector fragment follows a specific system as follows:

[0146] Target gene DNA fragment: 2μL

[0147] DNA fragment of the vector: 2 μL

[0148] 10×T4 DNALigase Buffer: 1μL

[0149] T4 DNALigase: 0.5 μL

[0150] ddH2O: 4.5μL

[0151] Total volume: 10μL

[0152] Reaction conditions: 22℃, 120min.

[0153] 4) Transform the ligation product into E. coli using E. coli DH5α competent cells, following the method in step (3).

[0154] 5) PCR detection, the method is the same as step (4).

[0155] 6) The positive recombinant plasmids obtained by detection are identified by double enzyme digestion, and the method is the same as in step (6).

[0156] 2. Construction of the plant knockdown expression vector pFGC5941-GmERF5

[0157] (1) Primer design

[0158] F(SEQ ID NO.9):CCGCTCGAGGAGCCCGCCCAGGTGAAC

[0159] R(SEQ ID NO.10): CATGCCATGGGAGGAGGTGAGGACGCAG

[0160] F(SEQ ID NO.11):GCTCTAGAGAGCCCGCCCAGGTGAAC

[0161] R(SEQ ID NO.12): CGGGATCCGAGGAGGTGAGGACGCAG

[0162] (2) Construction of plant knockdown expression vector pFGC5941-GmERF5

[0163] The pMDTM18-T-GmERF5 plasmid obtained during the construction of the plant overexpression vector pCAMBIA3301-GmERF5 was PCR amplified to obtain the target fragment with the same restriction sites as the vector. After sequencing confirmed that there were no errors, it was ligated into the vector pFGC5941 in both directions.

[0164] 3. Preparation of Agrobacterium competent cells by CaCl2 method

[0165] (1) Pick a single colony of LBA4404 and put it into 10mLYEP medium (containing 25mg / LRif), and incubate overnight in a constant temperature shaker at 28℃ and 220rpm.

[0166] (2) Using a pipette, draw 800 μL of the bacterial culture for propagation in a clean bench and inoculate it a second time to activate it to OD. 600 It is approximately 0.6.

[0167] (3) Take an appropriate amount of bacterial solution and place it in an EP tube on an ice bath for 30 minutes, then centrifuge at 4000×g for 5 minutes at low temperature, and discard the waste liquid in the EP tube.

[0168] (4) Pre-cool the sterilized 0.05M CaCl2 solution at low temperature in advance, 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.

[0169] (5) Take an appropriate amount of CaCl2 solution in the clean bench and repeatedly aspirate to precipitate the bacterial cells. Then, dispense the competent cells and store them at -80℃.

[0170] 4. Freeze-thaw transformation of Agrobacterium

[0171] (1) Use a pipette to aspirate 3 μL of the recombinant plasmid and transfer it into 100 μL of LBA4404 competent cells, and mix gently.

[0172] (2) Place the mixture on ice for 5 minutes, in liquid nitrogen for 5 minutes, incubate in a water bath at 37°C for 5 minutes, and then immediately transfer it to an ice bath for 3 minutes.

[0173] (3) Take 800 μL of YEP culture medium in a clean bench and revive it for 4 hours in a constant temperature shaker at 28℃ and 150 rpm.

[0174] (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 at 28℃ upside down for 3 days.

[0175] 5. Identification of Agrobacterium transformants

[0176] Single colonies were picked and placed in YEP liquid medium (Rif, Kan), plasmids were extracted, and enzyme digestion was performed for identification.

[0177] The results are as follows Figure 3 and Figure 4 As shown:

[0178] The unmutated pMDTM18-T-GmERF5 plasmid and the empty pCAMBIA3301 vector plasmid were double-digested using Nco1 and Pml1 restriction endonucleases. After agarose gel electrophoresis, a vector fragment of approximately 10.3 kb and a gene fragment of approximately 600 bp were recovered. These fragments were ligated using T4-DNA ligase and transformed into *E. coli* DH5α to obtain a single colony of the recombinant plasmid pCAMBIA3301-GmERF5. The recombinant plasmid was then transformed into *Agrobacterium* LBA4404, and double-enzyme digestion confirmed that GmERF5 had been successfully ligated into the plant expression vector. Figure 3 ).

[0179] After designing specific primers and amplifying the target fragment by PCR, the fragments were ligated to the plant expression vector pFGC5941 in both directions, creating a hairpin structure in the transcription region. Then, the recombinant plasmid pFGC5941-ERF5, which was confirmed by enzyme digestion and sequencing analysis, was transformed into Agrobacterium LBA4404. Double enzyme digestion confirmed that the two target fragments were ligated to the plant expression vector pFGC5941 in both directions. Figure 4 ).

[0180] Example 3

[0181] Soybean genetic transformation

[0182] 1. Culture medium components

[0183] (1) Germination medium (GM)

[0184] Composition: 1.0 mg / L 6-BA + 0.8% agar + 2% sucrose + B5 salt, pH 5.8.

[0185] (2) Co-culture medium (CCM)

[0186] Liquid culture medium composition: 3% sucrose + B5 salt + 3.9 g / L MES + 0.1 mg / L GA3 + 1.67 mg / L 6-BA + 200 μMAS, pH 5.4.

[0187] Solid culture medium composition: 3% sucrose + B5 salt + 3.9g / L LMES + 200μMAS + 0.1mg / L GA3 + 1.67mg / L 6-BA + 0.8% agar + 0.158g / L Na2S2O3 + 0.154g / L LDTT + 1.0g / L L-Cys, pH 5.4.

[0188] (3) Recovery medium (SIM)

[0189] Composition: 0.8% agar + 3% sucrose + B5 salt + plant hormones (500mg / L Cef, 1.67mg / L 6-BA, 0.59g / L MES), pH 5.6.

[0190] (4) SEM (Sewing Medium)

[0191] Composition: 3% sucrose + B5 salt + 0.8% agar + plant hormones (100mg / L L-Pyr + 50mg / L L-Asp + 0.59g / L MES + 250mg / L Cef + 0.5mg / L GA3 + 0.1mg / L IAA + 1.0mg / L ZR), pH 5.6.

[0192] (5) Rooting medium (RM)

[0193] Composition: 0.8% agar + 2% sucrose + 1 mg / L IBA + 0.59 g / L LMES + B5 salt, pH 5.6.

[0194] 2. Agrobacterium cotyledon node transformation method and soybean plant regeneration

[0195] (1) Select high-quality "Dongnong 50" soybean seeds and sterilize them in a fume hood using chlorine sterilization for 16 hours.

[0196] (2) Sterile seeds were planted in germination medium and cultured in a tissue culture room at 25°C for 5 days.

[0197] (3) Remove the hypocotyl from the germinated sterile soybean seedlings, cut the soybeans longitudinally along the midline, remove the apical bud and axillary bud, and make 3 cuts at the cotyledon node.

[0198] (4) Shake the bacterial culture until OD 600 Centrifuge at 0.5, 12000 rpm, resuspend in the infection liquid, and place the prepared cotyledonary explants into it. Shake at 28℃, 120 rpm, for 30 min.

[0199] (5) Dry the bacterial solution from the infected cotyledonary explants, place them on the adaxial surface of CCM solid medium, arrange them neatly, and culture them in the dark in the tissue culture room for 3 days.

[0200] (6) The cotyledon nodes were rinsed three times in sterile water, the liquid was absorbed with sterilized filter paper, and then inserted into the recovery solid culture medium and cultured in the tissue culture room for 15 days.

[0201] (7) Cut off the resistant buds that have grown to 1-2 cm and insert them into the elongation medium. Place them in the tissue culture room for 15 days.

[0202] (8) Once the elongated seedlings are healthy and robust, insert them into the rooting medium and culture them upright for 20 days.

[0203] (9) When the elongated seedlings have grown a suitable amount of roots, remove them and place them in humus soil mixed with vermiculite. Then, cultivate soybean conversion plants in a greenhouse.

[0204] Example 4

[0205] Detection and identification of GmERF5 genetically modified soybeans:

[0206] 1. PCR detection of GmERF5 transgenic soybean plants

[0207] (1) Extraction of soybean genomic DNA (SDS micro-method)

[0208] 1) Take 0.1g of leaves 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.

[0209] 2) Add 80 μL of 10% SDS and mix well.

[0210] 3) Bathe in a 65℃ water bath for 10 minutes, invert and mix well, repeat 3 times.

[0211] 4) Add 100 μL of 5M KAC, vortex for 6 seconds, and place on crushed ice for an ice bath for 30 minutes.

[0212] 5) Vortex for 20 seconds, add an equal volume of chloroform / isoamyl alcohol (24:1), and centrifuge at 12000 rpm for 15 min.

[0213] 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.

[0214] 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.

[0215] 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℃.

[0216] 2. RT-PCR detection of transgenic plants

[0217] (1) Primer design

[0218] Transformed plants were cultured in a greenhouse until stage V2. Leaf DNA was extracted using the SDS-PAGE method, and RT-PCR was used to detect whether the transformed plants carried the exogenous gene bar. Primer sequences were as follows:

[0219] F(SEQ ID NO.13):ATATCCGAGCGCCTCGTGCAT

[0220] R(SEQ ID NO.14):GGTCTGCACCATCGTCAACCACT

[0221] (2) PCR detection of the bar gene.

[0222] (3) The PCR amplification products were detected by agarose gel electrophoresis.

[0223] 2. Western blot analysis of GmERF5-overexpressing transgenic soybean plants

[0224] GmERF5-overexpressing transgenic soybean plants that tested positive with bar primers were subjected to Western blot detection of the GmERF5-myc fusion protein using an anti-myc antibody. 0.2 g of leaves from the bar-positive plants 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).

[0225] (1) Preparation of main solutions:

[0226] 1) Electrophoresis buffer: 25mM Tris, 0.25M glycine, 0.1% SDS;

[0227] 2) Transfer buffer: 25mM Tris, 0.25M glycine, 0.1% SDS, 20% methanol;

[0228] 3) TBS buffer: 100mM Tris-HCl pH=7.5, 150mM NaCl;

[0229] 4) TBST buffer: TBS buffer containing 0.05% Tween 20;

[0230] 5) Blocking solution: TBST buffer containing 5% skim milk powder.

[0231] (2) SDS-PAGE electrophoresis

[0232] 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.

[0233] (3) Transfer membrane

[0234] 1) Prepare a PVDF membrane of the same size as the adhesive and immerse the PVDF membrane in methanol for 3-5 minutes.

[0235] 2) Immerse two sponge pads, two 3mm filter papers, a glass rod, and the clamps for membrane transfer into the transfer solution.

[0236] 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.

[0237] 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.

[0238] (4) Protein immune response

[0239] 1) Transfer the PVDF membrane to the blocking solution and shake it at room temperature for 1 hour to seal it.

[0240] 2) Transfer the PVDF membrane to a TBST solution containing primary antibodies (Anti-myc, abcam; ab9108, diluted 5000 times) and incubate at room temperature for 1 hour.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] 6) Use filter paper to absorb the liquid on the membrane.

[0245] (5) Chemiluminescence

[0246] 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.

[0247] 2) The membrane was placed in a chemiluminescence imaging system (Tianneng 5500) for analysis.

[0248] 3. Southern blotting detection of GmERF5 transgenic soybean plants that interfered with the test

[0249] Follow the instructions in the DIG High Prime DNA Labeling and Detection Starter Kit.

[0250] (1) DIG probe labeling (random primer method)

[0251] 1) Using the PCAMBIA3301 plasmid containing the bar gene as a template, the bar gene was amplified by PCR, and the gel was recovered and the volume was adjusted to 16 μL with sterile water.

[0252] 2) After boiling water bath for 10 min, ice bath for 10 min, add 4 μL of solution No. 1, mix well, and label the reaction conditions as follows: 37℃, 20 h; and terminate the reaction conditions as follows: 65℃, 10 min.

[0253] 3) Store at -20℃ for later use.

[0254] (2) Preparation of plant genomic DNA

[0255] The method for extracting genomic DNA is described in section 1.

[0256] (3) Enzymatic digestion and purification of genomic DNA

[0257] Enzyme digestion system:

[0258] 10×Tango Buffer: 20μL

[0259] HindIII: 10 μL

[0260] DNA 150: μL

[0261] RNase-free H2O: 20 μL

[0262] After mixing, incubate at 37°C for 16 hours.

[0263] Genomic DNA was purified according to the instructions of the OMGEA DNA purification kit.

[0264] (4) Gel treatment

[0265] 1) Perform electrophoresis on the enzyme-digested genomic DNA and plasmids using a 1% agarose gel at 25V overnight.

[0266] 2) When the DNA has spread to about two-thirds of the gel, remove the gel, cut off the excess gel, and place it in a lunchbox.

[0267] 3) Add 200 mL of denaturing solution (enough to cover the gel), shake at room temperature for 30 min, replace with a new 200 mL of denaturing solution, and shake at room temperature for another 30 min.

[0268] 4) Remove the denaturing solution and rinse once with sterile ultrapure water.

[0269] 5) Add 200 mL of neutralization solution (enough to cover the gel), shake at room temperature for 30 min, replace with a new 200 mL of neutralization solution, and shake at room temperature for another 30 min.

[0270] (5) Imprint

[0271] 1) Take a porcelain dish and add about 1L of 20×SSC solution.

[0272] 2) Place a glass plate on a porcelain dish, cut a 3mm piece of filter paper that is large enough to be submerged in the solution at both ends, and place it on the glass plate to allow the filter paper to fully absorb the liquid and remove air bubbles between the filter paper and the glass plate.

[0273] 3) Take out the neutralized gel and place it on a 3mm filter paper on a glass plate to remove air bubbles between the filter paper and the gel.

[0274] 4) Cut a piece of nylon membrane and two pieces of 3mm filter paper of the same size as the gel. Cover the gel with the nylon membrane soaked in 20×SSC solution. Then take a piece of 3mm filter paper that has been pretreated with 2×SSC solution, lay it flat on the nylon membrane, and remove air bubbles.

[0275] 5) Cover the 3mm filter paper with absorbent paper of the same size, place a weight on the absorbent paper to make close contact, and use the siphon method to make the DNA migrate upward. Replace the absorbent paper in time and keep it for 16-20 hours.

[0276] 6) Place the transferred nylon membrane with the DNA imprinted side facing up and incubate at 1200J for 3 minutes for UV crosslinking.

[0277] (6) Prehybridization and hybridization

[0278] 1) Place the UV-crosslinked nylon membrane into a hybridization bottle with the imprinted side inside, add an appropriate amount of solution No. 7, preheat solution No. 7 to 42°C to remove air bubbles, and then pre-hybridize in a hybridization furnace at 42°C for 2 hours at a speed of 20 rpm.

[0279] 2) Add new solution No. 7 containing 10 μL of probe to the hybridization bottle, place it in the hybridization oven, and hybridize for 20 h.

[0280] (7) Washing and developing

[0281] 1) Remove the hybridization membrane from the hybridization bottle, place it in a porcelain dish, add 100 mL of washing solution I, shake at room temperature for 10 min, replace with new washing solution I and wash for another 10 min, then remove washing solution I.

[0282] 2) Add 100 mL of washing solution II, shake and wash at 65°C for 15 min, remove washing solution II, and repeat once.

[0283] 3) Add 100 mL of freshly prepared 1×Blocking solution and shake on a shaker for 60 min to remove the 1×Blocking solution.

[0284] 4) Add 10 mL of Antibody solution, shake for 30 min, and remove the Antibody solution.

[0285] 5) Add 100 mL of washing buffer solution, shake at room temperature for 15 min, remove the washing buffer, and repeat once.

[0286] 6) Add 20 mL of detection buffer solution and let stand at room temperature for 2-5 min to remove the detection buffer.

[0287] 7) Blot the nylon membrane dry, place it in a hybridization bag, add 1 mL of substrate solution (solution No. 5), let it stand at room temperature in the dark for 10 min, and then incubate it at 37℃ for 10 min.

[0288] 8) In a darkroom, after development and fixing, the hybridization result is obtained.

[0289] (8) Main solution components

[0290] Denaturing solution: 1.5M NaCl, 0.5M NaOH.

[0291] Neutralization solution (pH=8.0): 0.5M Tris-HCl, 1.5M NaCl.

[0292] 20×SSC (pH=7.0): 3M NaCl, 0.3M sodium citrate.

[0293] Maleic acidbuffer (PH=7.5): 0.15M NaCl, 0.1M Maleic acid.

[0294] Detectionbuffer (PH=9.5): 0.1M Tris-HCl, 0.1M NaCl.

[0295] Washing solution I: 50ml 20×SSC (pH=7.0), 5mL 10% SDS, bring to a final volume of 500mL.

[0296] Washing solution II: 12.5ml 20×SSC (pH=7.0), 5mL 10% SDS, bring the volume to 500mL.

[0297] 1×Blocking solution: Mix 20 mL of solution 6 and 180 mL of malachic acid buffer (pH = 7.5).

[0298] Washing buffer: Maleic acid buffer containing 3% Tween 20 (pH=7.5).

[0299] The results are as follows Figure 5-9 As shown:

[0300] DNA was extracted from T3 generation GmERF5 overexpressing transgenic plants using the SDS-PAGE method. Using these plants as templates, the exogenous gene bar was detected by RT-PCR, yielding bar gene-positive plants. Figure 5 Further analysis using Western blot revealed three GmERF5-OE transgenic plants. Figure 6 ).

[0301] DNA was extracted from T3 generation GmERF5-RNAi transgenic plants using the SDS-PAGE method. Using these plants as templates, the exogenous gene bar was detected by RT-PCR, yielding bar gene-positive plants. Figure 7 Further analysis using Southern blot revealed three GmERF5-RNAi transgenic plants. Figure 8 ).

[0302] qRT-PCR analysis confirmed that the expression level of GmERF5 in T3 GmERF5-OE transgenic soybean plants was significantly higher than that in wild-type plants, and the expression level in T3 GmERF5-RNAi transgenic soybean plants was significantly lower than that in wild-type plants. Figure 9 ).

[0303] Example 5

[0304] Analysis of drought resistance in GmERF5 transgenic soybean plants:

[0305] 1. Preparation of test materials

[0306] GmERF5-OE and GmERF5-RNAi transgenic soybean T3 generation seeds and "Dongnong 50" (WT) were planted in a 1:1 vermiculite:soil mixture and placed in a photothermal incubator with complete water retention and irrigation for 21 days (14-hour photoperiod, 25°C, 60% relative humidity). Irrigation was then stopped for 8 days to induce drought stress in 21-day-old GmERF5-OE, GmERF5-RNAi transgenic soybean and wild-type soybean seedlings. The drought-resistant phenotype of the transgenic lines was observed and photographed. Then, wilted plants were rehydrated to restore growth, and the drought-resistant phenotype of the transgenic soybeans was recorded 2 days later.

[0307] 2. Measurement of pore size

[0308] 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.

[0309] 3. Determination of superoxide dismutase activity

[0310] 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.

[0311] Place 0.1g of sample in a 1.5mL EP tube, add 1mL of crude enzyme extract, and grind into a homogenate using a cryogenic grinder. Centrifuge at 14000rpm, 4℃ for 10min, and transfer the supernatant to a new 1.5mL EP tube as the test sample. Following the kit instructions, mix 100μL of reagent 3 with 4.9mL of distilled water, denoted as solution A1; mix 50mL of reagent 1 with 250μL of reagent 2, denoted as solution A2; dissolve 100μL of reagent 4 in 5mL of water, denoted as solution A3. In a 1mL cuvette, add 50μL of the test sample (50μL of distilled water for the control group) (sample V), 50μL of solution A1, 800μL of solution A2, and 100μL of solution A3 sequentially, making the total reaction volume 1mL. Mix well and let stand for 30min. Measure the absorbance at 450nm using a spectrophotometer, denoted as control A and assay A.

[0312] 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).

[0313] The activity of SOD in the sample was calculated using the following formula:

[0314] Inhibition percentage (P) = [(Control A - Assay A) / Control A] × 100%

[0315] SOD activity (U / mgprot) = [P / (1-P)×Vtotal] / (Vsample×Cpr)

[0316] 4. Determination of peroxidase activity

[0317] 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.

[0318] Place 0.1g of sample in a 1.5mL EP tube, homogenize with 1mL of crude enzyme extract at low temperature, centrifuge at 14000rpm and 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 the working solution. According to the kit instructions, add 50μL of the test sample (V sample) and 950μL of the working solution to a 1mL cuvette and mix well (V total reaction). Use a spectrophotometer to measure the absorbance A1 at 470nm for 1min and the absorbance A2 at 2min, i.e., the reaction time is 1min.

[0319] The protein concentration (Cpr) of the sample was determined according to the instructions of the BCA Protein Assay Kit (Beyotime).

[0320] The activity of POD in the sample is calculated using the following formula:

[0321] POD(U / mgprot)=[(A2-A1)×Vtotal / (Vsample×Cpr)]÷0.01 / T

[0322] Experimental results: such as Figure 10-14 As shown.

[0323] Under normal watering conditions (WW), the growth of WT, GmERF5-OE, and GmERF5-RNAi transgenic plants was consistent with no significant differences. After 4 days of drought treatment (D1), the opposite leaves of GmERF5-OE transgenic plants showed slight wilting, while the trifoliate leaves remained unchanged. The opposite leaves of WT plants showed moderate to severe wilting, and the trifoliate leaves showed slight wilting. The opposite true leaves of GmERF5-RNAi transgenic plants showed severe wilting, and the trifoliate leaves showed moderate wilting. After 8 days of treatment (D2), the leaves of GmERF5-OE plants showed moderate to severe wilting. The opposite true leaves of WT plants showed severe wilting, and the growing point showed moderate shrinkage. All leaves of GmERF5-RNAi transgenic plants showed wilting, and the shoot tip growing point showed severe shrinkage. Two days after rehydration (RW), the growth points of GmERF5-RNAi transgenic plants and wild-type plants died and they did not recover growth, while the growth points of GmERF5-OE plants were healthy and the plants recovered growth. Figure 10 The above results demonstrate that overexpression of GmERF5 can improve the drought resistance of soybean plants.

[0324] To further investigate the drought resistance mechanism of GmERF5, the stomatal status of GmERF5-OE and GmERF5-RNAi transgenic plants and WT plants in the WW, D1, D2, and RW groups was observed. Figure 11 and Figure 12 As shown, under moderate drought treatments (D1) and severe drought treatments (D2), the stomatal closure degree of GmERF5-OE transgenic plants was significantly higher than that of WT plants, while the stomatal closure degree of GmERF5-RNAi transgenic plants was significantly lower than that of WT plants. These results indicate that under drought stress, GmERF5 can reduce water loss by decreasing stomatal pore size, thereby alleviating leaf wilting and maintaining a higher relative water content.

[0325] like Figure 13 As shown: There was no significant difference in SOD activity among GmERF5-OE, WT, and GmERF5-RNAi transgenic plants in the WW group; the SOD activity of GmERF5-OE transgenic plants in the D1 and D2 groups was significantly higher than that of WT (*P<0.05), while the SOD activity of GmERF5-RNAi transgenic plants was significantly lower than that of WT (*P<0.05); there was no significant difference in SOD activity among GmERF5-OE, WT, and GmERF5-RNAi transgenic plants in the RW group. These results indicate that overexpression of GmERF5 under drought stress can enhance the drought tolerance of soybean by increasing the activity of SOD enzymes in soybean plants.

[0326] like Figure 14As shown: There was no significant difference in POD activity among GmERF5-OE, WT, and GmERF5-RNAi transgenic plants in the WW group; the POD activity of GmERF5-OE transgenic plants in groups D1 and D2 was significantly higher than that of WT plants (**P<0.01), while the POD activity of GmERF5-RNAi transgenic plants was significantly lower than that of WT plants (*P<0.05); there was no significant difference in SOD activity among GmERF5-OE, WT, and GmERF5-RNAi transgenic plants in the RW group. These results indicate that overexpression of GmERF5 under drought stress can enhance the drought tolerance of soybean by increasing the activities of SOD and POD enzymes in soybean plants.

[0327] Example 6

[0328] Salt tolerance analysis of GmERF5 transgenic soybean plants:

[0329] 1. Transgenic soybean seeds of WT, GmERF5-OE and GmERF5-RNAi were planted in vermiculite and irrigated with water containing 0 mM NaCl, 150 mM NaCl, 250 mM NaCl and 350 mM NaCl respectively. They were then placed in a light and temperature incubator for 3 days (photoperiod 14 h, temperature 25℃, relative humidity 60%) and their germination and root length were observed.

[0330] 2. To further determine the relevant physiological indicators in the transgenic plants, WT, GmERF5-OE, and GmERF5-RNAi transgenic soybean plants were planted in a 1:1 vermiculite:soil mixture and placed in a light-temperature incubator with complete water retention for 21 days (14-hour photoperiod, 25°C, and 60% relative humidity). The 21-day-old WT, GmERF5-OE, and GmERF5-RNAi transgenic soybean seedlings were irrigated with 0 mM NaCl and 250 mM NaCl water, respectively. After 24 hours, samples were taken to determine the activities of their antioxidant enzymes SOD and POD.

[0331] The results are as follows Figure 15 and 16 As shown: There was no significant difference in the germination status of GmERF5-OE, GmERF5-RNAi, and WT under 0 mM NaCl treatment; the radicle length of GmERF5-OE transgenic soybean seeds was significantly longer than that of WT seeds under 150 mM NaCl and 250 mM NaCl treatment (**P<0.01); while under 350 mM NaCl treatment, only GmERF5-OE transgenic soybean seeds showed slight germination. These results indicate that overexpression of GmERF5 can improve the salt tolerance of transgenic soybean seeds.

[0332] 21-day-old transgenic soybean plants and WT plants were irrigated with 250 mM NaCl, and the activities of SOD and POD were measured. Figure 17 and 18 As shown: After NaCl treatment, the SOD activity of GmERF5-OE transgenic plants was significantly higher than that of WT plants (**P<0.01), while the SOD activity of GmERF5-RNAi transgenic plants was significantly lower than that of WT plants (*P<0.05); the POD activity of GmERF5-OE transgenic plants was significantly higher than that of WT plants (**P<0.01), while the POD activity of GmERF5-RNAi transgenic plants was significantly lower than that of WT plants (*P<0.05). These results indicate that overexpression of GmERF5 can enhance antioxidant enzyme activity and thus improve the salt tolerance of GmERF5-OE transgenic soybean plants.

[0333] Example 7

[0334] Analysis of GmPOD1 expression levels in GmERF5 transgenic soybean plants under drought and salt stress:

[0335] For specific instructions, please refer to Examples 1, 5, and 6.

[0336] Experimental results: such as Figure 19 As shown.

[0337] Depend on Figure 19 As shown, after drought and 250 mM NaCl treatment, the expression level of GmPOD1 in GmERF5-OE transgenic soybean plants was significantly higher than that in WT and GmERF5-RNAi (**P<0.01). In conclusion, overexpression of GmERF5 can increase the expression level of GmPOD1 and enhance POD activity to improve the drought resistance and salt tolerance of soybeans.

[0338] As can be seen from the above embodiments, the overexpression of the GmERF5 gene in this invention enhances the antioxidant enzyme activity of transgenic soybean plants, thereby increasing the tolerance of soybeans to drought and salt stress, and providing a new regulatory gene resource for improving the drought resistance and salt tolerance of soybeans.

[0339] 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 a GmERF5 gene in altering the drought resistance and / or salt tolerance of soybeans, wherein overexpression of the GmERF5 gene in soybeans enhances the drought resistance and / or salt tolerance of soybeans; The nucleotide sequence of the GmERF5 gene is shown in SEQ ID NO.1; The amino acid sequence of the protein encoded by the GmERF5 gene is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The method for overexpressing the GmERF5 gene includes the following steps: The GmERF5 gene was cloned into the pCAMBIA3301 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 overexpression of the GmERF5 gene.

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