CN115873085B - Application of soybean gene GmMAX2a in plant stress resistance - Google Patents

Abstract

The invention discloses the application of soybean gene GmMAX2a in plant stress resistance, and belongs to the technical field of plant breeding. In order to improve the stress resistance of plants, especially salt and alkali tolerance. The invention provides a soybean gene GmMAX2a and the application of the soybean gene in plant stress resistance, mainly resistance to NaCl stress and alkali stress. It provides efficient and scientific technical means for mining genes with significant functions and transgenic molecular breeding.

Description

Application of soybean gene GmMAX2a in plant stress resistance

Technical Field

The invention belongs to the technical field of plant breeding, and particularly relates to application of a soybean gene GmMAX2a in plant stress resistance.

Background Art

How to improve the alkali tolerance of crops is a major problem that needs to be solved in agricultural production. In recent years, with the continuous development of molecular biology, the use of increasingly mature genetic engineering technology to cultivate new crop varieties with excellent traits and good stress tolerance has become one of the important means of modern crop improvement. With the rapid development of cutting-edge disciplines such as modern molecular biology, bioinformatics, genetic engineering, genomics and proteomics, efficient and scientific technical means have been provided for the exploration of genes with significant functions and transgenic molecular breeding.

Summary of the invention

The purpose of the invention is to improve the stress resistance of plants, especially salt resistance and alkali resistance.

The present invention provides a soybean protein GmMAX2a, and the amino acid sequence of the GmMAX2a is shown in SEQ ID NO.4.

It is further defined that the nucleotide sequence of the gene encoding soybean protein GmMAX2a is shown in SEQ ID NO.3.

The invention discloses a vector containing the above coding gene.

The present invention provides an application of the soybean protein GmMAX2a, the encoding gene or the vector in plant stress resistance.

It is further defined that the plant stress resistance is resistance to NaCl stress and resistance to alkali stress.

It is further defined that the resistance to NaCl stress is to subject the Arabidopsis seedlings to salt stress treatment under 75-125 mmol/L NaCl for 7 days; the resistance to alkali stress is to subject the Arabidopsis seedlings to alkali stress treatment under 0.5-0.7 mmol/L NaHCO ₃ for 7 days.

It is further defined that the plant is a monocotyledonous plant and/or a dicotyledonous plant.

It is further defined that the dicotyledonous plant may specifically be a leguminous plant and/or a cruciferous plant and/or an Asteraceae plant; the leguminous plant may be soybean, Lotus japonica, alfalfa or Pongamia chinensis; the cruciferous plant may be Arabidopsis thaliana or rapeseed; the Asteraceae plant may be sunflower; and the Arabidopsis thaliana may be Arabidopsis thaliana (Columbia ecotype col-0).

The present invention provides a method for improving plant stress resistance, and the specific steps of the method are as follows:

Step 1: Connect the gene sequence shown in SEQ ID NO.3 with the pCAMBIA1300 vector to obtain a recombinant vector;

Step 2: Transform the recombinant vector obtained in step 1 into Agrobacterium to obtain recombinant Agrobacterium;

Step 3: Infect plants with the recombinant Agrobacterium obtained in step 2 to obtain transgenic plants.

It is further defined that the Agrobacterium in step 2 is Agrobacterium tumefaciens GV3101.

It is further defined that the primers for cloning the gene sequence shown in SEQ ID NO.3 in step 1 are shown in SEQ ID NO.1 and SEQ ID NO.2.

Beneficial effects: The present invention discovered a soybean salt-alkali tolerance gene GmMAX2a, and conducted tissue localization analysis on it, and found that its expression level in young stems was significantly higher than that in other tissues. The gene expression pattern of the GmMAX2a gene was analyzed under salt and alkali stress. Under these three abiotic stresses, the GmMAX2a gene responded to varying degrees in soybean leaves and roots, and the relative expression of the gene was regulated by the time of the stress, indicating that the GmMAX2a gene played a certain role in the process of soybean responding to salt, alkali, and stress. The GmMAX2a gene was subjected to GUS staining analysis, and the expression of the GmMAX2a gene was induced by salt and alkali stress, and GUS signals could be detected at various time points. The experiments of the present invention have shown that overexpressing the gene in Arabidopsis can enhance the tolerance of Arabidopsis to salt and alkali stress in the seedling stage, indicating that the gene can lay a foundation for the research on breeding transgenic plants with salt and alkali tolerance. The expression of salt- and alkali-related marker genes in transgenic Arabidopsis was analyzed. It was found that under salt and alkali conditions, stress marker genes such as RD29B, COR15A, and RD29A were upregulated in GmMAX2a-overexpressing transgenic Arabidopsis, and the expression levels were significantly higher than those in WT. The GmMAX2a gene may enhance tolerance to salt and alkali stress by regulating the expression levels of stress-induced marker genes.

Salt stress resistance is specifically resistance to NaCl stress, which is reflected in that under the condition of NaCl stress: the root length of the transgenic plant is longer than that of the recipient plant, and the fresh weight of the transgenic plant is higher than that of the recipient plant. The alkali stress resistance is specifically resistance to NaHCO ₃ stress, which is reflected in that under the condition of NaHCO ₃ stress: the root length of the transgenic plant is longer than that of the recipient plant, and the fresh weight of the transgenic plant is higher than that of the recipient plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is an expression analysis of the GmMAX2a gene in different soybean tissues;

Figure 2 shows the expression levels of the GmMAX2a gene at different time points under salt and alkali stress; 200mmol/L NaCl: A-leaf, B-root; 50mmol/L NaHCO ₃ : C-leaf, D-root;

Figure 3 shows GUS staining of the GmMAX2a gene promoter under different stresses; A: 125mmol/L NaCl; B: 50mmol/LNaHCO ₃ ;

FIG4 is a molecular identification of GmMAX2a gene-transfected Arabidopsis plants;

FIG5 shows the phenotype, root length and fresh weight of GmMAX2a transgenic Arabidopsis thaliana under salt stress;

FIG6 shows the phenotype, root length and fresh weight of GmMAX2a transgenic Arabidopsis under alkaline stress;

Figure 7 is the expression analysis of Marker genes related to salt stress;

Figure 8 is an analysis of alkaline stress-related Marker gene expression;

DETAILED DESCRIPTION

Example 1. Cloning of soybean GmMAX2a gene

1. Processing of plant materials

Select full soybean Dongnong 50 seeds and sterilize them in a solution containing 6% sodium hypochlorite for 10 minutes, pour out the sodium hypochlorite, rinse with sterile water 3 to 4 times, place on moist filter paper, and culture in the dark at 25℃ for 4 days to germinate. When the buds grow to about 1 to 2 cm, transfer them to a pot filled with Hogland culture solution, fix them with space cotton, immerse the buds in the culture solution, and place them in a greenhouse for culture. The growth conditions in the greenhouse are 25℃/20℃ (day/night) and the photoperiod is 16 hours of light/8 hours of darkness. When the seedlings grow to 3 weeks old, take their leaves and put them in EP tubes and store them at -80℃.

2. RNA Extraction

Total RNA was extracted from the leaves of the 3-week-old soybean seedlings using the Plant Total RNA Isolation Kit (Foregene).

3. Obtaining cDNA

The total RNA was used as a template for reverse transcription to obtain cDNA.

4. PCR amplification

Using the above cDNA as a template, Primer-KS and Primer-KAS primers were used for PCR amplification to obtain PCR amplification products. The primer sequences are as follows:

Primer-KS: 5’-ACGATGATAAGGGCGGTACCAATATCTTACCGGAGAATGGGC-3’ (SEQ IDNO.1);

Primer-KAS: 5'-AGGCTACGTAGGATCCTTACATGTCAATCACATATTCGACG-3' (SEQ ID NO. 2).

PCR amplification system (50 μl): cDNA 1 μl, Primer-F 1 μl, Primer-R 1 μl, Prime Star Mix 12.5 μl, ddH ₂ O 9.5 μl.

PCR amplification conditions: 98°C for 10 s, 52°C for 10 s, 72°C for 2 min 40 s, 35 cycles; 72°C for 5 min; terminate the reaction at 4°C.

The PCR amplification product was subjected to 1% agarose gel electrophoresis to obtain a band with a molecular weight of approximately 2Kb, and the PCR amplification product was recovered using an agarose gel extraction kit (Omega Gel Extraction kit); it was connected to the pCAMBIA-1300 vector to obtain a recombinant plasmid, which was named pCAMBIA1300-GmMAX2a, and was transformed into Escherichia coli DH5α competent cells and then sent for sequencing.

The sequencing results showed that the PCR amplification obtained an amplification product of 2157 bp in size, which was named GmMAX2a gene, and its nucleotide sequence was shown in SEQ ID NO. 3. The amino acid sequence of GmMAX2a was shown in SEQ ID NO. 4.

ATGGGCGACGGCAGCATCGTGGGCCATCTCCCGGAGGAGATTCTGTTGAACGTGT TCGCGGCGGTTTCCGACACCCGCACGCGGAACGCGTTGTCGTTGGTATCGTGGAGCTTCTACCGGTTGGAGCGCAAGACGCGCACATCTCTCACGCTCCGCGGCAACGCGCGTGAC CTCCACCTCATCCCGACCTCCTTCAAGCACGTCACGCACCTCGACCTCCTTCCTCTCGCCGTGGGGCCACGCGCTCTTCT GTCCTCCTCCTCCTCCGCCGCCGCCGCCGCCGTCG ACCACCAGCGACACCTCGCGCAACATCTCCGCGCCGCGTTCCCGCGCGTCACCTCACTCGCCGTCTACGCGCGTGACCCGGACACTCTCCGCCTCCTCCTCCTCTCCCCATGGCCGG AGCTCTCCGCCGTCAAGCTCGTCCGGTGGCACCAGCGCCCGCCGACCTCCGCGAACGAAGCCGACTTCGCTGAGCTCTTCAAGAAGTGCCGATCGCTCGCCTCCCTCGACCTCTCCT CCTACTACCACTGGACGGAGGACATCCCGACGGTGCTCGCCGCAAACCCTATCTCCGCCGCCTTTCTCCGCCGCCTCAACCTCCTAACAACCTCCTTAACAGAAGGATTCAAGTCTCACGAAATCGAATCGATCACCGCGTCG TGCCCTAACCTGGAGCACTTTCTCGCGGTCTGC AACTTCGATCGGAGATATAGGGTCCGTCAGCGACGACACGCTGGTTTCTATTGCTTCCAATTGCCCAAAGCTATCGTTGCTTCACTTGGCCGATACGTCGTCGTTTTCAAGTCGCAG AGAGGAGGACGAGGGTTTCGACGGAGAAGACGCTAGCATTAGCCGCGCTGCTCTTATGACTTTGTTTCCTGGACTTCCTTTTGGAAGAGCTCGTGTTAGATGTTTGTAAAAATGTC AGAGAGAGTAGTTTCGCTTTTGAAGTGGTGGGTTCAAAGTGCCCTAATTTGAGGGTTCTAAAATTGGGACAGTTTCAAGGGATTTGCTTGGCCTTTGAGTCTCGGCTTGACGGAATTG CCCTTTGCCACGGGC TTCAATCGTTGTCTGTTGGTAACTGCGCGGACCTTGATGACATGGGGTTAATTGAGATTGCCAGGGGGTGTTCGAGACTGGTTAGATTTGAGCTTCAGGGTTG CAGGCTTGTGACGGAGCGTGGACTGAGGACCATGGCTTGTTTGCTTGGCAGGACTCTGATTGATGTCAGGGTTTCTTGCTGCGTAAACCTTGACACAGCTGCGACTTCCAGGCTTT GGAGCCAATTCGCGAGCAGATTGAGCGGCTCCATGTGGATTGTGTGTGGAATGGGTTGAAGGAGAGTGATGGCCTGGGACATGGGTTTCTTAGTTTTGATTTGAATGGTTTGGATGAACAGGATGATGTGGGTA AACTCATGGATTACTACTTTGGGGGTGGAGAATGTGAGAAC ACAAGCAAAAGGAAGAGGCAGAGATGCGAGTATCAAATGAGGGTTCATGATTCCTTTT TGGAAAGCAATGGCAATGGTTTCTATGGCAAGAGCTGGGACAAGCTGCAGTATCTTTCTCCTGGATAAAAGTTGGTGATCTCTTGACTCCATTGCCGGTGGCAGGGTTGGAAGATTG TCCCGTCTTGGAAGAGATTCGGATTAAGGTTGAAGGAGATTGTAGGGGGCAGCCAAAGCCAGCAGAGAGCGAATTTGGTCTCAGCATTCTGGCTTGTTATCCTCAGCTATTGAAGAT GCAGCTGGACTGTGGTGAT ACTAAAGGTTATGCTCTCACGGCACCCTCTGGGCAAATGGATTTGAGCCTGTGGGAGAGGTTTCTTCTGAATGGCATTGGCAGTTTGAGTCTCAGTGAGCTTCATTACTGGCCACCACAAGATGAGGATGTGAACCAGAGGAGTGTGTCACTTCCAGCTGCTGGCTTGCTACAAGAATGTTACACTTTGAGAAAGCTCTTCATTCACGGAACAGCA CATGAACATTTCATGAACTTTTTTCTTAAGATACAAAACCTTAGGGATGTACAGCTGAGAGAAGATTATTATCCAGCTCCTGAAAATGACATGAGTACAGAGATGAGGGTAGGTTCGT GCAGCCGCTTTGAAGATGCACTGAATAGGCGTCGAATATGTGATTGA (SEQ ID NO. 3)

MGDGSIVGHLPEEILLNVFAAVSDTRTRNALSLVSWSFYRLERKTRTSLTLRGNARDL HLIPTSFKHVTHLDLSFLSPWGHALFCSSSSSAAAAAVDHQRHLAQHLRAAFPRVTSLAVYARDPDTLRLLLLSPWPELSAVKLVRWHQRPPTSANEADFAELFKKCRSLASLDLSSYYHW TEDIPTVLAANPISAAFLRRLNLLTTSLTEGFKSHEIESITASCPNLEHF LAVCNFDRRYIGSVSDDTLVSIASNCPKLSLLHLADTSSFSSRREEDEGFDGEDASISRAALMTLFSGLPLLEELVL DVCKNVRESSFAFEVVGSKCPNLRVLKLGQFQGICLAFESRLDGIALCHGLQSLSVGNCADLDDMGLIEIARGCSRLVRFELQGCRLVTERGLRTMACLLGRTLIDVRVSCCVNLDTAATLR ALEPIREQIERLHVDCVWNGLKESDGLGHGFLSFDLNGLDEQDDVGKLMDYYFGGGECENTSKRKRQRCEYQMRVHDSFLESNGNGFYGKSWDKLQYLSLWIKVGDLLTPLPVAGLED CPVLEEIRIKVEGDCRGQPKPAESEFGLSILACYPQLLKMQLDCGDTKGYALTAPSGQMDL SLWERFLLNGIGSLSLSELHYWPPQD EDVNQRSVSLPAAGLLQECYTLRKLFIHGTAHEHFMNFFLKIQNLRDVQLREDYYPAPENDMSTEMRVGSCSRFEDALNRRRRICD(SEQ ID NO.4)

Example 2. Analysis of expression characteristics of soybean GmMAX2a gene

1. Relative expression of GmMAX2a gene in different soybean tissues

1. Processing of plant materials

Soybean seeds were sterilized with a solution containing 6% sodium hypochlorite for 10 minutes and germinated in a petri dish with distilled water for 4 days. The growing seedlings were fixed on the holes of the foam board and cultured in a black plastic container containing 1/2 Hoagland nutrient solution. The growth conditions in the greenhouse were 25°C/20°C (day/night) and the photoperiod was 16 hours of light/8 hours of darkness. Seedlings with good growth conditions were selected and moved into sterilized soil. When the seedlings grew to 3 months old, different tissues of wild soybean (including young leaves, young roots, young stems, roots, stems, leaves, flowers, and pods) were quickly taken and stored at -80°C.

2. Extraction of total RNA and acquisition of cDNA

The Plant Total RNA Isolation Kit (Foregene) was used to extract total RNA from different tissues of the wild soybean (including young leaves, young roots, young stems, roots, stems, leaves, flowers, and pods); total RNA was used as a template for reverse transcription to obtain cDNA.

3. Real-time PCR

Using the above cDNA as a template, Primer-qS and Primer-qAS primers were used to detect the expression of the GmMAX2a gene by real-time PCR. The primer sequences are as follows:

Primer-qS: 5’-TCTTCATTCACGGAACAGCA-3’ (SEQ ID NO.5);

Primer-qAS: 5'-TCAATCACATATTCGACGCCT-3' (SEQ ID NO. 6).

Real-time PCR reaction conditions: 94°C 10 min→[94°C 30 s→64°C 30 s]×40→60°C 30 s→72°C 10 min→94°C 2 min.

Real-time PCR uses the comparative CT method ( ^ΔΔ CT) to calculate gene expression, with GmGAPDH as the internal reference gene and untreated samples as the control. The difference in target gene expression is expressed by the multiple of the treated sample relative to the untreated sample at each time point. Each sample includes 3 biological replicates and 3 technical replicates. The data is the average of 3 biological replicates. If one value has a large deviation, the average of the two data is taken. The original data is standardized. The standardized data is analyzed for significant differences by T-test. Relative expression calculation method: ^2-ΔΔ CT = ^2- ( ^Δ CT treatment - ^Δ CT control) = ^2- [(CT treatment target gene - CT treatment internal reference gene) - (CT control target gene - CT control internal reference gene)]. The primer sequence of the internal reference gene is as follows:

GsGAPDH S: 5'-GACTGGTATGGCATTCCGTGT-3' (SEQ ID NO. 7);

GsGAPDH AS: 5'-GCCCTCTAGTTCCTCCTTGA-3' (SEQ ID NO. 8).

The results are shown in Figure 1: GmMAX2a gene is expressed in all tissues of soybean, and the expression level is relatively high in young stems, and relatively low in young roots and young leaves. There is no significant difference in the expression level in roots, stems, leaves, flowers, and pods at the mature stage, indicating that the expression of GmMAX2a gene in soybean has temporal and spatial specificity.

2. Analysis of the expression pattern of GmMAX2a gene in soybean roots and leaves at different times under alkaline stress

1. Processing of plant materials

Soybean seeds were sterilized with a solution containing 6% sodium hypochlorite for 10 min and germinated in a petri dish with distilled water for 4 days. The growing seedlings were fixed on the holes of the foam board and cultured in a black plastic container containing 1/2 Hoagland nutrient solution. The growth conditions in the greenhouse were 25℃/20℃ (day/night) and the photoperiod was 16 hours of light/8 hours of darkness. When the soybean seedlings grew to 18 days old, they were subjected to salt stress under 200mmol/L NaCl and alkaline stress under 50mM NaHCO ₃ (pH 8.5). Three soybean plants were selected at 0h, 1h, 3h, 6h, 12h, and 24h, and their leaves and root tips of 3cm were cut as tissue samples to be tested, quickly frozen in liquid nitrogen, and then stored at -80℃.

2. Extraction of total RNA and acquisition of cDNA

The total RNA of the tissue samples to be tested obtained in step 1 after being treated for different time periods was extracted using the Plant Total RNA Isolation Kit (Foregene). The total RNA was used as a template for reverse transcription to obtain cDNA.

3. Real-time PCR

Using the above cDNA as a template, Primer-qS and Primer-qAS primers were used to detect the expression of the GmMAX2a gene by real-time PCR. The primer sequences are as follows:

Primer-qS: 5’-TCTTCATTCACGGAACAGCA-3’;

Primer-qAS: 5’-TCAATCACATATTCGACGCCT-3’.

Real-time PCR reaction conditions: 94°C 10 min→[94°C 30 s→64°C 30 s]×40→60°C 30 s→72°C 10 min→94°C 2 min.

Real-time PCR uses the comparative CT method (ΔΔCT) to calculate gene expression, with the wild soybean GsGAPDH gene as the internal reference gene and the untreated sample as the control. The difference in target gene expression is expressed by the multiple of the treated sample relative to the untreated sample at each time point. Each sample includes 3 biological replicates and 3 technical replicates. The data is the average of 3 biological replicates. If one value has a large deviation, the average of the two data is taken. The original data is standardized. The standardized data is analyzed for significant differences by T-test. Relative expression calculation method: 2 ^-ΔΔCT = 2-(ΔCT treatment-ΔCT control) = 2-[(CT treatment target gene-CT treatment internal reference gene)-(CT control target gene-CT control internal reference gene)]. The primer sequence of the internal reference gene is as follows:

GsGAPDH S: 5'-GACTGGTATGGCATTCCGTGT-3';

GsGAPDH AS: 5'-GCCCTCTAGTTCCTCCTTGA-3'.

The results are shown in Figure 2: Under these three abiotic stresses, the GmMAX2a gene responded to varying degrees in soybean leaves and roots, and the relative expression level of the gene was regulated by the time of the stress, indicating that the GmMAX2a gene plays a certain role in the soybean response to salt and alkali stress.

3. GUS staining analysis of tissue expression sites of GmMAX2a gene in Arabidopsis

1. Processing of plant materials

Select full soybean Dongnong 50 seeds and sterilize them in a solution containing 6% sodium hypochlorite for 10 minutes, pour out the sodium hypochlorite, rinse with sterile water 3 to 4 times, place on moist filter paper, and culture in the dark at 25°C for 4 days to germinate. When the buds grow to about 1 to 2 cm, transfer them to a pot filled with Hoagland culture solution, fix them with space cotton, immerse the buds in the culture solution, and place them in a greenhouse for culture. The growth conditions in the greenhouse are 25°C/20°C (day/night) and the photoperiod is 16 hours of light/8 hours of darkness. When the seedlings grow to 3 weeks old, take their leaves and put them in EP tubes and store them at -80°C.

2. Extraction of genomic DNA

The genomic DNA of the soybean roots was extracted according to the instructions of the Plant DNA Isolation Kit (Foregene).

3. Acquisition of GmMAX2a-pro gene

The genomic DNA of the soybean root was used as a template and PCR amplification was performed using primers GmMAX2a-pS and GmMAX2a-pAS to obtain a PCR amplification product, namely GmMAX2a-pro (containing the promoter sequence of the GmMAX2a gene).

GmMAX2a-pS: 5'-CGGGATCCAAGGCCAACAGTAAAGACAGGGAC-3' (SEQ ID NO. 9);

GmMAX2a-pAS: 5'-CATGCCATGGTATCCGGTAAGATATTTCCCAATTC-3' (SEQ ID NO. 10).

PCR amplification system (50 μl): cDNA 1 μl, Primer-F 1 μl, Primer-R 1 μl, Prime Star Mix 12.5 μl, ddH2O 9.5 μl.

PCR amplification conditions: 98°C for 10 s, 60°C for 10 s, 72°C for 2 min 40 s, 35 cycles; 72°C for 5 min; terminate the reaction at 4°C.

The PCR amplification product was subjected to 1% agarose gel electrophoresis to obtain a band with a molecular weight of about 2.2Kb, and the PCR amplification product was recovered using an agarose gel recovery kit (Omega Gel Extraction kit). The recovered fragment (PCR amplification product) was connected to pCAMBIA-3301, and the connection product was transformed into Escherichia coli DH5α competent cells. Positive clones were selected based on the kanamycin resistance marker on the pCAMBIA-3301 vector and sent to the company for sequencing.

The sequencing results showed that: the PCR amplification obtained an amplification product of 2200 bp in size, which was named GmMAX2a-pro gene, and its nucleotide sequence was shown in SEQ ID NO.11.

TAAAAAAAACTAAGCTTTAAGTAGTTATTTAAAAGGTTTTCCCATTATCTTGTTATT GTTTTAGTGTTTTAATGATAGCAAATATGTTGATATTTTAATGACTTTTAAAAAATTAATGTTATAATATACCTGTTTTGAAGACATTAATTTTACAAAAGAAATTAATATAAAAATAAAAAAAATGGTTCAACACATAAATGAGTAATATATTAATACTTTATTTTTAAAAAAAGTACTTTATTTGTACCTAAAAATACTTTAGTTTTTTCAAAA GAATGCAATACTTATCCCTTAAAATTCT ACGAAAAGGCCAACAGTAAAGACAGGGACAATTGAATCCATCAAATGTATCTTGATTCTTGCCGGTAGCAGGCTCGTGCAAGGTGCAAATCAGCTAATGCGTTTGGATTTTTGTCAAC ACCTTATATATATATATATATATATATATATATATATATAAGTAAAACTTAAAATAATTTTTGGTCTAAGTGCAGGTTTCTAAACAATTTGGATAAGCGAGAATTTATAAATTTACTTT TAAAATAATTTCGTTTTATAAAAGTGGAGTTTAATTAAAAATAAAGTAAATTCTACTTGATTTTTTAAAGATTATCTTTACAATAAAATTGAATATGAAAATTTTAACTTGATATAAATTCAAA GTATATTTAAGTGGCGTTGGATTCGACTTTTTGGGATATTATTTTTTCAATGACAATAATAAGATTTTTTTAAATTACTTTACTATTTTTTATTTTTAATTAAAAAATT ATTTATATATTTAGGTTTTACAATATTATGTTTAACTTAGTCATGTTTAATTAAGAATGAACCACAAGGGTCTTCTAATTGTAGGAAATTTGAGTAGTAAGTTAAATGTCGTAAGTTCTAG CATCAGAGGTAACCTTTGTAATAATAATAATAATAATAATAATAATAAAAAATAGACAATGGGTTCGAAAATGCAAAATATAAGTCAGACGTTCTAAAGGCCAGCTAGAAT TAAATTATCAGTGTCTAATTATTAGTTTAAAGATCACATATAGAAACTAAATGAATTTTTTAAAAAAAAAAAATTATAAACAACGTTGTTAGTAGTACATTTTTATCATAGTCTTTAACAA AAATTATCTATTATTTTTCACTTATAAACTTTATTAAA TTGTAAACAAAATCGTTAACTAGTGATGCCAAGTAAGGCGTGCAACACTAATGTGTGTTCCAAATCCCAATCCCTGCCAAATATAGAAGCATTAGCCAAATAGAAAAAACCGCCCAAGATTCTAAAGAAGAAAAGGAAAG CCAAAAAAATAAAACTCTTCAAAATACTATTTCTTTTATAATACTTTTTATAATTCATTAAAATTTATGAAATATATAACATTATAAAAATATTAAATAAAAAGTAAAAGCTACACAATAATA TATTAAAACATTAAACTATTGCTTGATGTCGGCATGTCGCTGAACTAACAAGTGAAAATA GACTCAAATTTTATAAATAAATTCTCAATATTGAATTTTGTGATGATTAAAAGTGATTAATCAAATTTTAGAGGAGATATTATGTATCAATGAGTTAAAAAAATTATT TAAACTACTTTTCTAT AATAATATTAGTATGAAGTATCAAATTAATTAAAACCTTATAATAATAAAAACAAATTTGAAGTGAGTCAATATACAAGTATTGAATAGATTATGCGAAGTAGGAGGGAAGGGTGTTTAT GTAATGTGTATGGACCCATCCAATCCTGTATGGTTTGTCTGTATTAATTTTCCTTTTCCTTCATACGTGCAGAGAGATTTCGTAAGCACCATTAGACCCATTCCCTTGGCATGGCTATCTTTCACTAAAAACCAAACACACCCTAATTTTCCATTCTCTCTAAACTAAACTTTCCTTCCATATCGTATTCACGTGGGTCTGCATGCCCCCAAAAACCCCTCCCAAAAACTATCCATT AATATTTAATACCATCCCTCTCCTTACCCTAATTTA ACATCATACACCCAATCCATTTCCTCTCTTTCTCTAGAGTAAGAGTATCATACCACTCAGCTCAAAAATTTAATAAAAAAAAATA GAGATTTTTTTTTTTAATTTTATAATTTTGGTGAAAATAGTAAGAGATAGAGAGGTGGGAATTGGGAAATATCTTACCGGAGA (SEQ ID NO. 11)

4. Obtaining the recombinant vector pCAMBIA3301-GmMAX2aPromoter:GUS

The GmMAX2a-pro gene shown in SEQ ID NO.11 was inserted between the BamH I and Nco I restriction sites of the pCAMBIA3301 vector to obtain the recombinant vector pCAMBIA3301-GmMAX2aPromoter:GUS, which was then sequenced for verification.

The sequencing results showed that the recombinant vector pCAMBIA3301-GmMAX2aPromoter:GUS was obtained by replacing the DNA sequence between the BamH I and Nco I restriction sites of the pCAMBIA3301 vector with the GmMAX2a-pro gene shown in SEQ ID NO.11, while keeping the other sequences of the pCAMBIA3301 vector unchanged.

5. Obtaining transgenic Arabidopsis

The above recombinant vector pCAMBIA3301-GmMAX2aPromoter:GUS was transformed into Agrobacterium GV3101, and Arabidopsis thaliana was infected by the inflorescence infiltration method to obtain a transgenic strain.

6. Salt and alkali stress treatment

The transgenic Arabidopsis seedlings at the six-leaf stage obtained in step 5 were subjected to salt (125mmol/L NaCl) and alkali (50mmol/L NaHCO ₃ ) stress treatments, and the transgenic Arabidopsis seedlings were taken at 0h, 1h, 3h, 6h, and 12h, and stained with a staining solution containing X-Gluc.

The results are shown in Figure 3: No GUS signal was detected in transgenic Arabidopsis without stress (CK). At 12h of salt stress (125mmol/L NaCl), GUS staining was deepest in the axils of the leaves and petiole base, and there was also deep staining in the roots, at which time the expression of the GmMAX2a gene was the highest; secondly, at 1h and 6h of salt stress, GUS signals were also detected, and staining was mainly concentrated in the leaves and petioles (Figure 3-A). At 3h of alkali stress (50mmol/L NaHCO ₃ ), the color of Arabidopsis leaves, axils and stems was significantly darker, but there was almost no GUS signal expression in the roots; at 1h, GUS expression was mainly in the stems; there was no significant difference in staining at 6h and 12h of alkali stress. The above results show that the expression of the GmMAX2a gene is induced by salt and alkali stress, and GUS signals can be detected at all time points.

Example 3. Obtaining GmMAX2a-transgenic Arabidopsis plants and analyzing their phenotypes under salt and alkali stress

1. Obtaining GmMAX2a-transgenic Arabidopsis plants

1. Acquisition of GmMAX2a gene

Using pCAMBIA1300-GmMAX2a obtained in step 4 of Example 1 as a template, PCR amplification was performed using primers Primer-ES and Primer-EAS to obtain a PCR amplification product, namely the GmMAX2a gene. The primer sequences are as follows:

Primer-ES: 5’-ACGATGATAAGGGCGGTACCAATATCTTACCGGAGAATGGGC-3’;

Primer-EAS: 5’-AGGCTACGTAGGATCCTTACATGTCAATCACATATTCGACG-3’.

PCR amplification system (50 μl): cDNA 1 μl, Primer-F 1 μl, Primer-R 1 μl, Prime Star Mix 12.5 μl, ddH ₂ O 9.5 μl.

PCR amplification conditions: 98°C for 10 s, 52°C for 10 s, 72°C for 2 min 40 s, 35 cycles; 72°C for 5 min; terminate the reaction at 4°C.

2. Obtaining plant expression vector

The pCAMBIA 1300 vector and the PCR amplification product were digested with restriction endonucleases and ligated to obtain the recombinant vector pCAMBIA 1300-GmMAX2a, which was then sequenced and verified.

The sequencing results showed that the recombinant vector pCAMBIA 1300-GmMAX2a was obtained by replacing the DNA fragment between the restriction sites of the pCAMBIA 1300 vector with the GmMAX2a gene shown in SEQ ID NO.3, while keeping the other sequences of the pCAMBIA1300 vector unchanged.

3. Conversion

The recombinant vector pCAMBIA1300-GmMAX2a was transformed into Agrobacterium tumefaciens GV3101 by freeze-thaw method, and positive transformants (transformants containing the GmMAX2a gene shown in sequence 1 in the sequence table) were obtained by PCR identification and used to infect Arabidopsis plants.

4. Obtaining GmMAX2a-transgenic Arabidopsis

Agrobacterium containing the recombinant vector pCAMBIA 1300-GmMAX2a was used to infect wild-type Arabidopsis thaliana (Columbia ecotype col-0) by the Floral-dip method, and the infected Arabidopsis thaliana was cultured to obtain T ₀ generation GmMAX2a transgenic Arabidopsis thaliana seeds. After surface disinfection of T ₀ generation GmMAX2a transgenic Arabidopsis thaliana seeds, they were sown on 1/2MS medium containing 25 mg/L glufosinate-ammonium (Sigma, 45520) for screening to obtain T ₁ generation GmMAX2a transgenic Arabidopsis thaliana seedlings. This process was repeated until a T ₃ generation GmMAX2a transgenic Arabidopsis thaliana homozygous strain was obtained.

Extract genomic DNA from _T3 generation GmMAX2a transgenic Arabidopsis seedlings and perform RT-PCR identification. The specific steps are as follows:

The total RNA of the _T3 transgenic GmMAX2a Arabidopsis plants obtained in step 1 was extracted and reverse transcribed to obtain cDNA; using the cDNA as a template, the Primer-qS and Primer-qAS primer pairs, Actin S and Actin AS primer pairs were used to detect the expression of the GmMAX2a gene by RT-PCR to obtain PCR amplification products. The primer sequences are as follows:

Primer-qS: 5’-ACGATGATAAGGGCGGTACCAATATCTTACCGGAGAATGGGC-3’;

Primer-qAS: 5’-AGGCTACGTAGGATCCTTACATGTCAATCACATATTCGACG-3’.

Actin S: 5’-TTACCCGATGGGCAAGTC-3’;

Actin AS: 5’-GCTCATACGGTCAGCGATAC-3’.

PCR amplification system (50 μl): cDNA 1 μl, Primer-F 1 μl, Primer-R 1 μl, Prime Star Mix 12.5 μl, ddH ₂ O 9.5 μl.

PCR amplification conditions:

GmMAX2a: [98℃10s→52℃10s→72℃2min40s]×35→72℃5min→4℃ to terminate the reaction;

Actin 2: [98℃10s→52℃10s→72℃2min40s]×35→72℃5min→4℃ to terminate the reaction.

The PCR amplification products were subjected to 1% agarose gel electrophoresis, and the test results are shown in Figure 4: There was no amplification product in the RT-PCR of the wild-type Arabidopsis plants, while the _T3 generation GmMAX2a-transformed Arabidopsis homozygous strain # ₅ and the _T3 generation GmMAX2a-transformed Arabidopsis homozygous strain #8 could amplify the target band, indicating that the exogenous gene GmMAX2a gene has not only been successfully integrated into the Arabidopsis genome, but can also be transcribed and expressed normally in transgenic Arabidopsis. The T3 generation GmMAX2a-transformed Arabidopsis homozygous strain #5 and the _T3 generation GmMAX2a-transformed Arabidopsis homozygous strain #8 were selected for the next phenotypic analysis.

2. Phenotypic analysis of transgenic GmMAX2a Arabidopsis plants under salt and alkaline stress

1. Seedling phenotype and root length of transgenic GmMAX2a Arabidopsis under salt and alkali treatments

Select plump seeds of wild-type Arabidopsis thaliana (Columbia ecotype), _T3 generation GmMAX2a transgenic Arabidopsis thaliana homozygous line #5 and _T3 generation GmMAX2a transgenic Arabidopsis thaliana homozygous line #8, disinfect with 5% sodium hypochlorite disinfectant for 10 minutes, vernalize at 4℃ for 3 days, and sow on 1/2MS solid medium. After 5 days, select GmMAX2a transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana seedlings with consistent growth and place them horizontally in plates containing different concentrations of NaCl (75mmol/L, 100mmol/L, 125mmol/L) and different concentrations of NaHCO ₃ (0.5mmol/L, 0.6mmol/L, 0.7mmol/L) stress medium, grow vertically, observe and record the fresh weight (every 10 seedlings are weighed once) and root length of Arabidopsis thaliana in each treatment group and non-treatment group after 7 days, all experiments are repeated 3 times for each technical and biological replicates. Ten plants of each strain were tested in each experiment.

The results are shown in Figures 5 and 6: Under normal conditions (i.e., no stress and no treatment, control), there was no significant difference in the growth and development of the _T3 generation GmMAX2a-transgenic Arabidopsis homozygous line #5 and the _T3 generation GmMAX2a-transgenic Arabidopsis homozygous line #8 and the wild-type Arabidopsis, indicating that the introduced GmMAX2a gene did not affect the growth and development of Arabidopsis at the seedling stage.

After adding 75mmol/L, 100mmol/L, and 125mmol/L NaCl to the culture medium, the growth of the three strains was inhibited to varying degrees. Under 75mmol/L NaCl stress, the root length of transgenic strains #5 and #8 was significantly longer than that of WT, and the leaves were larger and darker in color. Under 125mmol/L NaCl stress, WT showed cotyledon albinism, and its growth was severely inhibited, and the plant almost died. The growth of transgenic strains was also significantly inhibited, but the overall growth condition was better than WT. The statistical results of root length and fresh weight showed that the main root length and fresh weight of transgenic strains #5 and #8 seedlings under salt stress were higher than those of WT, indicating that the GmMAX2a gene may be involved in the plant's response to salt stress, making it more adaptable to salt stress.

After being stressed in the medium containing 0.5mmol/L, 0.6mmol/L, and 0.7mmol/L NaHCO ₃ , the growth of the three strains showed differences, and the growth difference was most obvious in the 0.5mmol/L NaHCO ₃ medium. The root growth of WT, transgenic strains #5, and #8 in the 0.7mmol/L NaHCO ₃ medium was greatly affected, mainly manifested in the inhibition of lateral root growth and wilting of leaves. The statistical results of root length and fresh weight were similar to those of salt stress. The main root length and fresh weight of transgenic strains #5 and #8 were higher than those of WT. The above results show that the GmMAX2a gene can alleviate the damage of alkaline stress to the growth and development of transgenic Arabidopsis seedlings, but with the increase of NaHCO ₃ concentration, the growth inhibition of each strain is strengthened, and the difference in root length and fresh weight between WT and the two overexpression strains gradually narrows.

Example 4. Determination of the expression of marker genes related to salt and alkaline stress in transgenic Arabidopsis

1. Processing of plant materials

The plant material GmMAX2a transgenic overexpressing Arabidopsis thaliana line #8 was cut and used as the tissue sample to be tested, which was quickly frozen in liquid nitrogen and then stored at -80°C.

2. Extraction of total RNA and acquisition of cDNA

The total RNA of the tissue samples to be tested obtained in step 1 after being treated for different time periods was extracted using the Plant Total RNA Isolation Kit (Foregene). The total RNA was used as a template for reverse transcription to obtain cDNA.

3. Real-time PCR

Using the above cDNA as a template and using salt and alkali stress-related marker gene primers, the expression level of the GmMAX2a gene was detected by Real-time PCR. The primer sequences are as follows:

Salt stress-related marker genes: RD29B, SOS1, NXH1, AtRD22, KIN1, and COR15A.

RD29B-S: 5’-TGAAGGAGACGCAACAAGGG-3’ (SEQ ID NO. 12);

RD29B-AS: 5'-CAACGGTGGTGCCAAGTGAT-3' (SEQ ID NO. 13).

SOS1-S: 5’-CGGCAGCATGGTTAATGTGTAC-3’ (SEQ ID NO. 14);

SOS1-AS: 5'-TTGGCTGAAACGAGACCTTGA-3 (SEQ ID NO. 15)'.

NXH1-S: 5’-CCACTCGAACCGTGCATTACT-3’ (SEQ ID NO. 16);

NXH1-AS: 5'-CTCAAGCCTTACTAAGATCAGGAGG-3' (SEQ ID NO. 17).

AtRD22-S: 5’-CCCATTCGCGGTGTTCTACT-3’ (SEQ ID NO. 18);

AtRD22-AS: 5’-CCAAGTGGTTTGGGTTCCAA-3’ (SEQ ID NO. 19);

KIN1-S: 5’-AACAAGAATGCCTTCCAAGC-3’ (SEQ ID NO. 20);

KIN1-AS: 5’-CGCATCCGATACACTCTTTC-3’ (SEQ ID NO. 21);

COR15A-S: 5’-AATTTCAAGCACTTAAACTCGT-3’ (SEQ ID NO. 22);

COR15A-AS: 5’-AGAATGTGACGGTGACTGTG-3’ (SEQ ID NO. 23);

Alkaline stress-related marker genes: RD29A, COR47, COR15A, KIN1, H+-APase, and NADP-ME.

RD29A-S: 5’-GGCGTAACAGGTAAACCTAGAG-3’ (SEQ ID NO. 24);

RD29A-AS: 5’-TCCGATGTAAACGTCGTCC-3’ (SEQ ID NO. 25);

COR47-S: 5’-GGAGTACAAGAACAACGTTCCCGA-3’ (SEQ ID NO. 26);

COR47-AS: 5’-TGTCGTCGCTGGTGATTCCTCT-3’ (SEQ ID NO. 27);

COR15A-S: 5’-AATTTCAAGCACTTAAACTCGT-3’ (SEQ ID NO. 28);

COR15A-AS: 5’-AGAATGTGACGGTGACTGTG-3’ (SEQ ID NO. 29);

KIN1-S: 5’-AACAAGAATGCCTTCCAAGC-3’ (SEQ ID NO. 30);

KIN1-AS: 5’-CGCATCCGATACACTCTTTCC-3’ (SEQ ID NO. 31);

H+-APase-S: 5’-TTTGGATTATAAACCTCACTATATG-3’ (SEQ ID NO. 32);

H+-APase-AS: 5’-CCAGTCATTCCAACAATATGC-3’ (SEQ ID NO. 33);

NADP-ME-S: 5’-TGGTCTGATCTACCCGCCATT-3’ (SEQ ID NO. 34);

NADP-ME-AS: 5’-CGCCAATCCGAGGTCATAGG-3’ (SEQ ID NO. 35);

Real-time PCR reaction conditions: 94°C 10 min→[94°C 30 s→64°C 30 s]×40→60°C 30 s→72°C 10 min→94°C 2 min.

Real-time PCR uses the comparative CT method ( ^ΔΔ CT) to calculate gene expression, with wild soybean GsGAPDH gene as the internal reference gene and untreated samples as the control. The difference in target gene expression is expressed by the multiple of the treated sample relative to the untreated sample at each time point. Each sample includes 3 biological replicates and 3 technical replicates. The data is the average of 3 biological replicates. If one value has a large deviation, the average of the two data is taken. The original data is standardized. The standardized data is analyzed for significant differences by T-test. Relative expression calculation method: ^2-ΔΔ CT = ^2- ( ^Δ CT treatment - ^Δ CT control) = ^2- [(CT treatment target gene - CT treatment internal reference gene) - (CT control target gene - CT control internal reference gene)]. The primer sequence of the internal reference gene is as follows:

Actin2-S: 5’-TTACCCGATGGGCAAGTC-3’;

Actin2-AS: 5’-GCTCATACGGTCAGCGATAC-3’.

The results are shown in Figures 7 and 8: Under salt stress, the relative expression levels of the six marker genes in GmMAX2a overexpressing transgenic Arabidopsis were higher than those at 0h between 3-6h, and the expression levels in GmMAX2a overexpressing transgenic Arabidopsis were significantly higher than those in WT. Among them, the relative expression levels of RD29B, SOS1, NXH1, AtRD22 and COR15A in GmMAX2a overexpressing transgenic Arabidopsis were higher than those in WT at 6h of stress, and the difference was extremely significant; the expression level of KIN1 was extremely significantly different after 3h of salt stress.

After 50mmol/L NaHCO ₃ treatment for 3h and 6h, the relative expression levels of 6 alkaline stress-related marker genes in WT and GmMAX2a transgenic Arabidopsis were analyzed. The relative expression levels of the 6 marker genes in GmMAX2a transgenic Arabidopsis were significantly higher than those at 0h. The relative expression levels of RD29A, COR15A, KIN1, H+-APase, and NADP-ME genes in GmMAX2a transgenic Arabidopsis reached extremely significant levels at 3h of alkali treatment, and the expression levels also showed a downward trend with the extension of treatment time, but they were all higher than WT; the expression level of COR47 was always higher than WT, and the relative expression levels of the genes at 3h and 6h of alkali treatment reached extremely significant levels.

The above results showed that under salt and alkali conditions, stress marker genes such as RD29B, COR15A, and RD29A were upregulated in GmMAX2a overexpressing transgenic Arabidopsis, and the expression levels were significantly higher than those in WT. Therefore, the GmMAX2a gene may enhance tolerance to salt and alkali stress by regulating the expression levels of stress-induced marker genes.

Fresh weight data under different stresses (g):

Table 1CK

WT #5 #8 CK-1 0.0504 0.0591 0.054 CK-2 0.0497 0.0518 0.0502 CK-3 0.0558 0.0521 0.0566 AVERAGE 0.051966667 0.054333333 0.0536

Alkaline stress:

Table 2 NaHCO ₃ 0.5 mM

WT #5 #8 NaHCO3-0.5-1 0.0446 0.059 0.0582 NaHCO3-0.5-2 0.0416 0.0535 0.053 NaHCO3-0.5-3 0.0430 0.0559 0.0552 AVERAGE 0.043066667 0.056133333 0.055466667

Table 3 NaHCO ₃ 0.6 mM

Table 4 NaHCO ₃ 0.7 mM

WT #5 #8 NaHCO3-0.7-1 0.0446 0.0373 0.0352 NaHCO3-0.7-2 0.0297 0.0367 0.0326 NaHCO3-0.7-3 0.0303 0.0368 0.0397 AVERAGE 0.034866667 0.036933333 0.035833333

Salt stress

Table 5: NaCl 75mM

WT #5 #8 NaCl-75-1 0.0257 0.027 0.0474 NaCl-75-2 0.0691 0.0782 0.05 NaCl-75-3 0.0358 0.0628 0.0628 AVERAGE 0.043533333 0.056 0.0534

Table 6 NaCl 100mM

WT #5 #8 NaCl-100-1 0.0244 0.0526 0.0399 NaCl-100-2 0.0258 0.0225 0.0325 NaCl-100-3 0.0257 0.038 0.0247 AVERAGE 0.0253 0.0377 0.032366667

Table 7 NaCl 125mM

WT #5 #8 NaCl-125-1 0.0208 0.0418 0.0321 NaCl-125-2 0.0191 0.035 0.0257 NaCl-125-3 0.0201 0.0298 0.0343 AVERAGE 0.02 0.035533333 0.0307

Root length data under different stresses (cm):

Table 11CK

Table 12

Table 13

Table 14

Table 15

Table 16

Claims (5)

1. Soybean proteinGmMAX2aThe application of a gene encoding soybean protein GmMAX2a or a vector containing the gene encoding soybean protein GmMAX2a in plant stress tolerance is characterized in that the amino acid sequence of the GmMAX2a is shown as SEQ ID NO. 4; the nucleotide sequence of the gene for encoding the soybean protein GmMAX2a is shown as SEQ ID NO. 3; the stress resistance of the plant is alkali stress resistance; the plant is dicotyledon.

2. The use according to claim 1, wherein the alkali stress resistance is between 0.5 and 0.7mmol/L NaHCO ₃ Arabidopsis seedlings were subjected to alkali stress treatment for 7 days under the conditions.

3. A method for improving alkali stress resistance of dicotyledonous plants, comprising the specific steps of:

step 1: connecting a gene sequence shown in SEQ ID NO.3 with a pCAMBIA1300 vector to obtain a recombinant vector;

step 2: transferring the recombinant vector obtained in the step 1 into agrobacterium to obtain recombinant agrobacterium;

step 3: and (3) infecting the plant with the recombinant agrobacterium obtained in the step (2) to obtain a transgenic plant.

4. A method according to claim 3, wherein the agrobacterium in step 2 is agrobacterium tumefaciens GV3101.

5. The method according to claim 3, wherein the primers for cloning the gene sequence shown in SEQ ID NO.3 in step 1 are shown in SEQ ID NO.1 and SEQ ID NO. 2.