CN115873085B - Application of soybean gene GmMAX2a in plant stress resistance - Google Patents
Application of soybean gene GmMAX2a in plant stress resistance Download PDFInfo
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Abstract
The invention discloses an application of a soybean gene GmMAX2a in plant stress resistance, and belongs to the technical field of plant breeding. In order to increase the stress resistance of plants, in particular the salt and alkali resistance. The invention provides a soybean gene GmMAX2a and application of the soybean gene in plant stress resistance, mainly resisting NaCl stress and alkali stress. Provides a high-efficiency scientific technical means for the excavation of genes and transgenic molecular breeding with remarkable functions.
Description
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
How to improve the alkali resistance of crops is a great problem to be solved in agricultural production. In recent years, with the continuous development of molecular biology, the cultivation of new varieties of crops with excellent properties and good stress tolerance by using genetic engineering technology which is mature gradually has become one of the important means for improving modern crops. Along with the rapid development of leading edge subjects such as modern molecular biology, bioinformatics, genetic engineering, genome, proteomics and the like, the method provides a high-efficiency and scientific technical means for excavating genes with remarkable functions and breeding transgenic molecules.
Disclosure of Invention
The invention aims to improve stress resistance of plants, in particular salt and alkali resistance.
The invention provides soybean protein GmMAX2a, and the amino acid sequence of the GmMAX2a is shown as SEQ ID NO. 4.
Further defined, the nucleotide sequence of the gene encoding soy protein GmMAX2a is shown as SEQ ID NO. 3.
The invention discloses a vector containing the coding gene.
The invention provides application of the soybean protein GmMAX2a, the coding gene or the vector in plant stress resistance.
Further defined, the plant stress tolerance is resistance to NaCl stress and alkali stress.
Further defined, the NaCl stress resistance is that Arabidopsis thaliana seedlings are subjected to salt stress treatment for 7 days under the condition of 75-125mmol/L NaCl; 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.
Further defined, the plant is a monocot and/or dicot.
Further defined, the dicotyledonous plant may be, in particular, a leguminous plant and/or a cruciferous plant and/or a compositae plant; the leguminous plant can be soybean, radix et rhizoma Barbati, herba Medicaginis or cortex et radix Populi; the cruciferous plant may be arabidopsis thaliana or brassica napus; the asteraceae plant may be sunflower; the Arabidopsis thaliana may be Arabidopsis thaliana (Col-0, columbia ecotype).
The invention provides a method for improving stress resistance of plants, which comprises the following specific steps:
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.
Further defined, the agrobacterium described in step 2 is agrobacterium tumefaciens GV3101.
Further defined, 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.
The beneficial effects are that: the invention discovers a soybean salt and alkali tolerance gene GmMAX2a, and performs tissue localization analysis on the soybean salt and alkali tolerance gene GmMAX2a, and discovers that the expression quantity of the soybean salt and alkali tolerance gene GmMAX2a in young stems is obviously higher than that of other tissues. The GmMAX2a gene is subjected to gene expression pattern analysis under salt and alkali stress, under the three abiotic stresses, the GmMAX2a gene has different degrees of response in soybean leaves and roots, and the relative expression quantity of the gene is regulated and controlled by the stress time, so that the GmMAX2a gene has a certain effect in the process of responding to salt, alkali and stress of soybean. GUS staining analysis is carried out on the GmMAX2a gene, the expression of the GmMAX2a gene is induced by salt and alkali stress, and GUS signals can be detected at all time points. Experiments prove that the gene is over-expressed in the arabidopsis thaliana, so that the tolerance of the seedling stage of the arabidopsis thaliana to the saline-alkali stress can be enhanced, and the gene can lay a foundation for the research of cultivating transgenic plants with the saline-alkali resistance. Analysis of transgenic arabidopsis salt and alkali stress related Marker gene expression shows that under salt and alkali conditions, the adverse stress Marker genes such as RD29B, COR15A, RD A are up-regulated in GmMAX2a over-expressed transgenic arabidopsis, the expression quantity is obviously higher than that of WT, and the GmMAX2a gene can enhance the tolerance to salt and alkali stress by regulating the expression level of the adverse stress induction Marker genes.
The salt stress resistance is specifically NaCl stress resistance,is expressed in the condition of NaCl stress: the transgenic plants have longer roots than the recipient plants and higher fresh weights than the recipient plants. The alkali stress resistance is especially NaHCO resistance 3 Stress, manifested as NaHCO 3 Under stress conditions: the transgenic plants have longer roots than the recipient plants and higher fresh weights than the recipient plants.
Drawings
FIG. 1 is an analysis of expression of the GmMAX2a gene in different tissues of soybean;
FIG. 2 shows the expression levels of 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;
FIG. 3 shows GUS staining of GmMAX2a gene promoter under different stresses; a:125mmol/L NaCl; b:50mmol/L NaHCO 3 ；
FIG. 4 is a molecular identification of Arabidopsis plants transformed with GmMAX2a gene;
FIG. 5 is a phenotype, root length and fresh weight of GmMAX2a transgenic Arabidopsis under salt stress;
FIG. 6 is a phenotype, root length and fresh weight of GmMAX2a transgenic Arabidopsis under alkali stress;
FIG. 7 is a salt stress related Marker gene expression analysis;
FIG. 8 is an alkali stress related Marker gene expression analysis;
Detailed Description
EXAMPLE 1 cloning of the Soybean GmMAX2a Gene
1. Treatment of plant material
Selecting plump soybean Dongnong 50 seeds, sterilizing in a solution containing 6% sodium hypochlorite for 10min, pouring out sodium hypochlorite, washing 3-4 times with sterile water, placing on wet filter paper, culturing in dark at 25 ℃ for 4d to accelerate germination, transferring the buds to a pot containing Hoagland culture solution when the buds grow to about 1-2 cm, fixing with space cotton, immersing the buds in the culture solution, and placing the buds in a greenhouse for culturing. The growth conditions of the greenhouse were 25 ℃/20 ℃ (day/night), photoperiod 16 hours light/8 hours darkness. When seedlings grow to 3 weeks old, taking leaves of the seedlings and placing the leaves into an EP tube, and storing the leaves at the temperature of minus 80 ℃.
2. RNA extraction
Total RNA from leaves of the 3-week-old soybean seedlings was extracted using a Plant Total RNA Isolation Kit kit (Foregene).
3. cDNA acquisition
And (3) reversely transcribing the total RNA serving as a template to obtain cDNA.
4. PCR amplification
And (3) carrying out PCR amplification by using the cDNA as a template and adopting Primer-KS and Primer-KAS primers to obtain a PCR amplification product. The primer sequences were as follows:
Primer-KS：5’-ACGATGATAAGGGCGGTACCAATATCTTACCGGAGAATGGGC-3’ (SEQ ID NO.1)；
Primer-KAS：5’-AGGCTACGTAGGATCCTTACATGTCAATCACATATTCGACG-3’(SEQ ID NO.2)。
PCR amplification System (50 μl): cDNA 1. Mu.l, primer-F1. Mu.l, primer-R1. Mu.l, prime Star Mix 12.5. Mu.l, ddH 2 O 9.5μl。
PCR amplification conditions: 98℃10s,52℃10s,72℃2min40s,35 cycles; 72 ℃ for 5min; the reaction was terminated at 4 ℃.
Detecting the PCR amplified product by 1% agarose gel electrophoresis to obtain a band with the molecular weight of about 2Kb, and recovering the PCR amplified product by using an agarose gel recovery kit (Omega Gel Extraction kit); the recombinant plasmid is connected with a pCAMBIA-1300 vector to obtain the recombinant plasmid, the recombinant plasmid is named as pCAMBIA1300-GmMAX2a, and the recombinant plasmid is transformed into escherichia coli DH5 alpha competent cells and then is subjected to sequencing.
Sequencing results showed that: the PCR amplification product with the size of 2157bp is obtained and named as GmMAX2a gene, and the nucleotide sequence of the gene is shown as SEQ ID NO. 3. The sequence of GmMAX2a amino acid is shown as SEQ ID NO. 4.
ATGGGCGACGGCAGCATCGTGGGCCATCTCCCGGAGGAGATTCTGTTGAACGTGT TCGCGGCGGTTTCCGACACCCGCACGCGGAACGCGTTGTCGTTGGTATCGTGGAGCTTCTACCGGTTGGAGCGCAAGACGCGCACATCTCTCACGCTCCGCGGCAACGCGCGTGAC CTCCACCTCATCCCGACCTCCTTCAAGCACGTCACGCACCTCGACCTCTCCTTCCTCTCGCCGTGGGGCCACGCGCTCTTCTGCTCCTCCTCCTCCTCCGCCGCCGCCGCCGCCGTCG ACCACCAGCGACACCTCGCGCAACATCTCCGCGCCGCGTTCCCGCGCGTCACCTCACTCGCCGTCTACGCGCGTGACCCGGACACTCTCCGCCTCCTCCTCCTCTCCCCATGGCCGG AGCTCTCCGCCGTCAAGCTCGTCCGGTGGCACCAGCGCCCGCCGACCTCCGCGAACGAAGCCGACTTCGCTGAGCTCTTCAAGAAGTGCCGATCGCTCGCCTCCCTCGACCTCTCCT CCTACTACCACTGGACGGAGGACATCCCGACGGTGCTCGCCGCAAACCCTATCTCCGC CGCCTTTCTCCGCCGCCTCAACCTCCTAACAACCTCCTTAACAGAAGGATTCAAGTCTCACGAAATCGAATCGATCACCGCGTCGTGCCCTAACCTGGAGCACTTTCTCGCGGTCTGC AACTTCGATCGGAGATATATAGGGTCCGTCAGCGACGACACGCTGGTTTCTATTGCTTCCAATTGCCCAAAGCTATCGTTGCTTCACTTGGCCGATACGTCGTCGTTTTCAAGTCGCAG AGAGGAGGACGAGGGTTTCGACGGAGAAGACGCTAGCATTAGCCGCGCTGCTCTTATGACTTTGTTCTCTGGACTTCCTCTTTTGGAAGAGCTCGTGTTAGATGTTTGTAAAAATGTC AGAGAGAGTAGTTTCGCTTTTGAAGTGGTGGGTTCAAAGTGCCCTAATTTGAGGGTTCTAAAATTGGGACAGTTTCAAGGGATTTGCTTGGCCTTTGAGTCTCGGCTTGACGGAATTG CCCTTTGCCACGGGCTTCAATCGTTGTCTGTTGGTAACTGCGCGGACCTTGATGACATGGGGTTAATTGAGATTGCCAGGGGGTGTTCGAGACTGGTTAGATTTGAGCTTCAGGGTTG CAGGCTTGTGACGGAGCGTGGACTGAGGACCATGGCTTGTTTGCTTGGCAGGACTCTGATTGATGTCAGGGTTTCTTGCTGCGTAAACCTTGACACAGCTGCGACTCTCAGGGCTTT GGAGCCAATTCGCGAGCAGATTGAGCGGCTCCATGTGGATTGTGTGTGGAATGGGTTGAAGGAGAGTGATGGCCTGGGACATGGGTTTCTTAGTTTTGATTTGAATGGTTTGGATGAACAGGATGATGTGGGTAAACTCATGGATTACTACTTTGGGGGTGGAGAATGTGAGAAC ACAAGCAAAAGGAAGAGGCAGAGATGCGAGTATCAAATGAGGGTTCATGATTCCTTTT TGGAAAGCAATGGCAATGGTTTCTATGGCAAGAGCTGGGACAAGCTGCAGTATCTTTCTCTCTGGATAAAAGTTGGTGATCTCTTGACTCCATTGCCGGTGGCAGGGTTGGAAGATTG TCCCGTCTTGGAAGAGATTCGGATTAAGGTTGAAGGAGATTGTAGGGGGCAGCCAAAGCCAGCAGAGAGCGAATTTGGTCTCAGCATTCTGGCTTGTTATCCTCAGCTATTGAAGAT GCAGCTGGACTGTGGTGATACTAAAGGTTATGCTCTCACGGCACCCTCTGGGCAAATGGATTTGAGCCTGTGGGAGAGGTTTCTTCTGAATGGCATTGGCAGTTTGAGTCTCAGTGAG CTTCATTACTGGCCACCACAAGATGAGGATGTGAACCAGAGGAGTGTGTCACTTCCAGCTGCTGGCTTGCTACAAGAATGTTACACTTTGAGAAAGCTCTTCATTCACGGAACAGCA CATGAACATTTCATGAACTTTTTTCTTAAGATACAAAACCTTAGGGATGTACAGCTGAGAGAAGATTATTATCCAGCTCCTGAAAATGACATGAGTACAGAGATGAGGGTAGGTTCGT GCAGCCGCTTTGAAGATGCACTGAATAGGCGTCGAATATGTGATTGA(SEQ ID NO.3)
MGDGSIVGHLPEEILLNVFAAVSDTRTRNALSLVSWSFYRLERKTRTSLTLRGNARDL HLIPTSFKHVTHLDLSFLSPWGHALFCSSSSSAAAAAVDHQRHLAQHLRAAFPRVTSLAVYARDPDTLRLLLLSPWPELSAVKLVRWHQRPPTSANEADFAELFKKCRSLASLDLSSYYHW TEDIPTVLAANPISAAFLRRLNLLTTSLTEGFKSHEIESITASCPNLEHFLAVCNFDRRYIGSVSDDTLVSIASNCPKLSLLHLADTSSFSSRREEDEGFDGEDASISRAALMTLFSGLPLLEELVL DVCKNVRESSFAFEVVGSKCPNLRVLKLGQFQGICLAFESRLDGIALCHGLQSLSVGNCADLDDMGLIEIARGCSRLVRFELQGCRLVTERGLRTMACLLGRTLIDVRVSCCVNLDTAATLR ALEPIREQIERLHVDCVWNGLKESDGLGHGFLSFDLNGLDEQDDVGKLMDYYFGGGECENTSKRKRQRCEYQMRVHDSFLESNGNGFYGKSWDKLQYLSLWIKVGDLLTPLPVAGLED CPVLEEIRIKVEGDCRGQPKPAESEFGLSILACYPQLLKMQLDCGDTKGYALTAPSGQMDL SLWERFLLNGIGSLSLSELHYWPPQDEDVNQRSVSLPAAGLLQECYTLRKLFIHGTAHEHFMNFFLKIQNLRDVQLREDYYPAPENDMSTEMRVGSCSRFEDALNRRRICD(SEQ ID NO.4)
Example 2 analysis of expression Properties of the Soybean GmMAX2a Gene
1. Relative expression level of GmMAX2a gene in different soybean tissues
1. Treatment of plant material
Soybean seeds were sterilized with a solution containing 6% sodium hypochlorite for 10min and germinated in distilled water in petri dishes for 4 days. The grown seedlings were fixed on the wells of foam plates and cultured in black plastic containers containing 1/2Hoagland nutrient solution. The growth conditions in the greenhouse were 25 ℃/20 ℃ (day/night), photoperiod 16 hours light/8 hours darkness. Selecting young plants with good growth condition, transferring into sterilized soil, quickly taking different tissues (including young leaves, young roots, young stems, roots, stems, leaves, flowers and pods) of wild soybean after young plants grow to 3 months of age, and respectively storing at-80deg.C.
2. Extraction of Total RNA and cDNA acquisition
Extracting total RNA of different tissues (including young leaves, young roots, young stems, roots, stems, leaves, flowers and pods) of the wild soybeans respectively by using a Plant Total RNA Isolation Kit kit (Foregene); the total RNA is used as a template, and cDNA is obtained through reverse transcription.
3、Real-time PCR
Using the cDNA as a template, the expression level of GmMAX2a gene was detected by Real-time PCR using Primer-qS and Primer-qAS primers. The primer sequences are shown below:
Primer-qS：5’-TCTTCATTCACGGAACAGCA-3’(SEQ ID NO.5)；
Primer-qAS：5’-TCAATCACATATTCGACGCCT-3’(SEQ ID NO.6)。
conditions for Real-time PCR reactions: 94℃for 10 min- & gt [94℃for 30 s- & gt 64℃for 30s ]. Times.40- & gt 60℃for 30 s- & gt 72℃for 10 min- & gt 94℃for 2min.
Real-time PCR adopts comparative CT method ΔΔ CT) to calculate the gene expression level, gmGAPDH is used as an internal reference gene, and an untreated sample is used as a control. The target gene expression differences are expressed as fold-over of the treated samples relative to the untreated samples at each time point. Each sample included 3 biological replicates and 3 technical replicates, the data averaged 3 biological replicates, and if there was a large variance in one value, the two data averaged. The original data is normalized. The data after normalization were subjected to a difference significance analysis by T-test. The relative expression amount calculating method comprises the following steps: 2-ΔΔ CT＝ 2- ( Δ CT treatment Δ CT control) = 2- [ (CT treatment objective Gene-CT treatment reference Gene) - (CT control objective Gene-CT control reference Gene)]. The primer sequences of the internal reference genes are shown 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 FIG. 1: the GmMAX2a gene is expressed in each tissue of soybean, the expression level in young stems is relatively high, and the expression level in young roots and young leaves is relatively low. The expression quantity in the roots, stems, leaves, flowers and pods in the mature period is not obviously different, which indicates that the expression of the GmMAX2a gene in soybean has space-time specificity.
2. Expression pattern analysis of GmMAX2a gene in soybean root and leaf at different time under alkali stress treatment
1. Treatment of plant material
Soybean seeds were sterilized with a solution containing 6% sodium hypochlorite for 10min and germinated in distilled water in petri dishes for 4 days. The grown seedlings were fixed on the wells of foam plates and cultured in black plastic containers containing 1/2Hoagland nutrient solution. The growth conditions in the greenhouse were 25 ℃/20 ℃ (day/night), photoperiod 16 hours light/8 hours darkness.After the soybean seedlings grow to 18 days old, subjecting the soybean seedlings to salt stress treatment under the condition of 200mmol/L NaCl, and treating the soybean seedlings with 50mM NaHCO 3 And (3) carrying out alkali stress treatment on soybean seedlings under the condition of pH 8.5, respectively selecting 3 plants of soybeans at each time point of 0h, 1h, 3h, 6h, 12h and 24h, cutting 3cm of leaves and root tips of the soybeans to be taken as tissue samples to be tested, rapidly freezing the tissue samples in liquid nitrogen, and then preserving the tissue samples at the temperature of minus 80 ℃.
2. Extraction of Total RNA and cDNA acquisition
Extracting total RNA of the tissue samples to be tested after different time treatments obtained in the step 1 by adopting a Plant Total RNA Isolation Kit kit (Foregene); the cDNA is obtained by reverse transcription using the obtained total RNA as a template.
3、Real-time PCR
Using the cDNA as a template, the expression level of GmMAX2a gene was detected by Real-time PCR using Primer-qS and Primer-qAS primers. The primer sequences are shown below:
Primer-qS：5’-TCTTCATTCACGGAACAGCA-3’；
Primer-qAS：5’-TCAATCACATATTCGACGCCT-3’。
conditions for Real-time PCR reactions: 94℃for 10 min- & gt [94℃for 30 s- & gt 64℃for 30s ]. Times.40- & gt 60℃for 30 s- & gt 72℃for 10 min- & gt 94℃for 2min.
The Real-time PCR was performed using comparative CT (ΔΔCT) to calculate the gene expression level, using the wild soybean GsGAPDH gene as a reference gene, and using an untreated sample as a control. The target gene expression differences are expressed as fold-over of the treated samples relative to the untreated samples at each time point. Each sample included 3 biological replicates and 3 technical replicates, the data averaged 3 biological replicates, and if there was a large variance in one value, the two data averaged. The original data is normalized. The data after normalization were subjected to a difference significance analysis by T-test. The relative expression amount calculating method comprises the following steps: 2 -ΔΔCT =2- (Δct treatment- Δct control) =2- [ (CT treatment objective gene-CT treatment reference gene) - (CT control objective gene-CT control reference gene)]. The primer sequences of the internal reference genes are shown as follows:
GsGAPDH S：5'-GACTGGTATGGCATTCCGTGT-3'；
GsGAPDH AS：5'-GCCCTCTAGTTCCTCCTTGA-3'。
the results are shown in FIG. 2: under the three abiotic stresses, the leaves and roots of the soybean of the GmMAX2a gene have different degrees of response, and the relative expression quantity of the gene is regulated and controlled by the stress time, so that the GmMAX2a gene has a certain effect in the process of responding to salt and alkali stress of the soybean.
3. GUS staining method for analyzing tissue expression part of GmMAX2a gene in arabidopsis thaliana
1. Treatment of plant material
Selecting plump soybean Dongnong 50 seeds, sterilizing in a solution containing 6% sodium hypochlorite for 10min, pouring out sodium hypochlorite, washing 3-4 times with sterile water, placing on wet filter paper, culturing in dark at 25 ℃ for 4d to accelerate germination, transferring the buds to a pot containing Hoagland culture solution when the buds grow to about 1-2 cm, fixing with space cotton, immersing the buds in the culture solution, and placing the buds in a greenhouse for culturing. The growth conditions of the greenhouse were 25 ℃/20 ℃ (day/night), photoperiod 16 hours light/8 hours darkness. When seedlings grow to 3 weeks old, taking leaves of the seedlings and placing the leaves into an EP tube, and storing the leaves at the temperature of minus 80 ℃.
2. Extraction of genomic DNA
Genomic DNA from the roots of the soybeans was extracted according to the procedure of Plant DNA Isolation Kit (Foregene) kit instructions.
3. Acquisition of the GmMAX2a-pro Gene
The genomic DNA of the soybean root is used as a template, and the PCR amplification product, namely GmMAX2a-pro (a promoter sequence containing GmMAX2a gene) is obtained by using GmMAX2a-pS and GmMAX2a-pAS primers for PCR amplification.
GmMAX2a-pS：5'-CGGGATCCAAGGCCAACAGTAAAGACAGGGAC-3'(SEQ ID NO.9)；
GmMAX2a-pAS：5'-CATGCCATGGTATCCGGTAAGATATTTCCCAATTC-3'(SEQ ID NO.10)。
PCR amplification System (50 μl): 1 μl of cDNA, 1 μl of Primer-F, 1 μl of Primer-R, 12.5 μl of Prime Star Mix, and 9.5 μl of ddH 2O.
PCR amplification conditions: 98℃10s,60℃10s,72℃2min40s,35 cycles; 72 ℃ for 5min; the reaction was terminated at 4 ℃.
The PCR amplified product was subjected to 1% agarose gel electrophoresis to obtain a band having a molecular weight of about 2.2Kb, and the PCR amplified product was recovered by using an agarose gel recovery kit (Omega Gel Extraction kit). The recovered fragment (PCR amplified product) was ligated with pCAMBIA-3301, and the ligation product was transformed into E.coli DH 5. Alpha. Competent cells, and positive clones were selected based on the kanamycin resistance marker on the pCAMBIA-3301 vector and submitted to sequencing by the company.
Sequencing results showed that: PCR amplification gives 2200bp amplification product, which is named GmMAX2a-pro gene, and the nucleotide sequence is shown in SEQ ID NO. 11.
TAAAAAAAACTAAGCTTTAAGTAGTTATTTAAAAGGTTTTCCCATTATCTTGTTATT GTTTTAGTGTTTTAATGATAGCAAATATGTTGATATTTTAATGACTTTTAAAAAATTAATGTTATAATATACCTGTTTTGAAGACATTAATTTTACAAAAGAAATTAATATAAAAATAAAAA AAATGGTTCAACACATAAATGAGTAATATATTAATACTTTATTTTTAAAAAAAGTACTTTATTTGTACCTAAAAATACTTTAGTTTTTTCAAAAGAATGCAATACTTATCCCTTAAAATTCT ACGAAAAGGCCAACAGTAAAGACAGGGACAATTGAATCCATCAAATGTATCTTGATTCTTGCCGGTAGCAGGCTCGTGCAAGGTGCAAATCAGCTAATGCGTTTGGATTTTTGTCAAC ACCTTATATATATATATATATATATATATATATATATATATATATAAGTAAAACTTAAAATAATTTTTGGTCTAAGTGCAGGTTTCTAAACAATTTGGATAAGCGAGAATTTATAAATTTACTTT TAAATAATTTCGTTTTATAAAGTGGAGTTTAATTAAAAATAAAGTAAATTCTACTTGATTTTTTAAAGATTATCTTTACAATAAAATTGAATATGAAAATTTTAACTTGATATAAATTAATAC TTTTCTTAATTCAAAGTATATTTAAGTGGCGTTGGATTCGACTTTTTGGGATATTATTTTTTCAATGACAATAATAAGATTTTTTTAAATTACTTTACTATTTTTTATTTTTAATTAAAAAATT ATTTATATATTTTAGGTTTACAATATTATGTTTAACTTAGTCATGTTTAATTAAGAATGAACCACAAGGGTCTTCTAATTGTAGGAAATTTGAGTAGTAAGTTAAATGTCGTAAGTTCTAG CATCAGAGGTAACCTTTGTAATAATAATAATAATAATAATAATAATAATAATAATAAAAAATAGACAATGGGTTCGAAAATGCAAAATATAAGTCAGACGTTCTAAAGGCCAGCTAGAAT TAAATTATCAGTGTCTAATTATTAGTTTAAAGATCACATATAGAAACTAAATGAATTTTTTAAAAAAAAAAAATTATAAACAACGTTGTTAGTAGTACATTTTTATCATAGTCTTTAACAA AAATTATCTATTATTTTTCACTTATAAACTTTATTAAATTGTAAACAAAATCGTTAACTAGTGATGCCAAGTAAGGCGTGCAACACTAATGTGTGTTCCAAATCCCAATCCCTGCCAAATA TAGAAGCATTAGCCAAATAGAAAAAACCGCCCAAGATTCTAAAGAAGAAAAGGAAAG CCAAAAAAATAAAACTCTTCAAAATACTATTTCTTTTATAATACTTTTTATATTCATTAAAATTTATGAAATATATAACATTATAAAAATATTAAATAAAAAGTAAAAGCTACACAATAATA TATTAAAACATTAAACTATTGCTTGATGTCGGCATGTCGCTGAACTAACAAGTGAAAATA GACTCAAATTTTATAAATAAATTCTCAATATTGAATTTTGTGATGATTAAAAGTGATTAATCAAATTTTAGAGGAGATATTATGTATCAATGAGTTAAAAAATATTTAAACTACTTTTCTAT AATAATATTAGTATGAAGTATCAAATTAATTAAAACCTTATAATAATAAAAACAAATTTGAAGTGAGTCAATATACAAGTATTGAATAGATTATGCGAAGTAGGAGGGAAGGGTGTTTAT GTAATGTGTATGGACCCATCCAATCCTGTATGGTTTGTCTGTATTAATTTTCCTTTTCCTTCATACGTGCAGAGAGATTTCGTAAGCACCATTAGACCCATTCCCTTGGCATGGCTATCTT TCACTAAAAACCAAACACACCCTAATTTTCCATTCTCTCTAAACTAAACTTTCCTTCCATATCGTATTCACGTGGGTCTGCATGCCCCCAACAAAAACCCCTCCCAAAAACTATCCATT AATATTTAATACCATCCCTCTCCTTACCCTAATTTAACATCATACACCCAATCCATTTTCCTCTCTTTCTCTAGAGTAAGAGTATCATACCACTCAGCTCAAAAATTTAATAAAAAAAAATA GAGATTTTTTTTTTTAATTTTATAATTTTGGTGAAAATAGTAAGAGATAGAGAGGTGGGAATTGGGAAATATCTTACCGGAGA(SEQ ID NO.11)
4. Acquisition of the recombinant vector pCAMBIA3301-GmMAX2aPromoter: GUS
The GmMAX2a-pro gene shown in SEQ ID No.11 is inserted between BamHI and Nco I cleavage sites of the pCAMBIA3301 vector to obtain a recombinant vector pCAMBIA3301-GmMAX2aPromoter: GUS. And sequence verification is performed on the same.
Sequencing results showed that: GUS is a vector obtained by replacing the DNA sequence between BamH I and Nco I cleavage sites of the pCAMBIA3301 vector with the GmMAX2a-pro gene shown in SEQ ID NO.11 and keeping other sequences of the pCAMBIA3301 vector unchanged.
5. Obtaining transgenic Arabidopsis thaliana
GUS is transformed into agrobacterium GV3101, and the arabidopsis thaliana is infected by an inflorescence infiltration method to obtain a transgenic strain.
6. Salt and alkali stress treatment
The transgenic Arabidopsis seedlings in six leaf stage obtained in the step 5 are respectively subjected to salt (125 mmol/L NaCl) and alkali (50 mmol/L NaHCO) 3 ) Stress treatment, transgenic Arabidopsis seedlings were taken at time points of 0h, 1h, 3h, 6h, and 12h, respectively, and X-G-containingThe luc staining solution stained it.
The results are shown in FIG. 3: in the absence of stress (CK), no GUS signal was detected in transgenic arabidopsis. In the case of salt stress (125 mmol/L NaCl) for 12 hours, GUS staining is deepest in leaves and axilla at the basal part of the petioles, and root parts are also deeply stained, so that the expression quantity of GmMAX2a genes is highest; secondly GUS signal was also detected at salt stress for 1h and 6h, staining was concentrated mainly on leaves and petioles (FIG. 3-A). Alkali stress (50 mmol/L NaHCO) 3 ) At 3h, the color of the arabidopsis leaves, axillary and stems is obviously darker, but the root has almost no GUS signal expression; whereas at 1h, the GUS expression site was predominantly on the stem; there was no significant difference in staining conditions for alkali stress for 6h and 12 h. The results show that the expression of GmMAX2a gene is induced by salt and alkali stress, and GUS signals can be detected at all time points.
Example 3 acquisition of GmMAX2 a-transformed Arabidopsis plants and phenotypic analysis under salt and alkali stress
1. Acquisition of Arabidopsis plants transformed with GmMAX2a
1. Acquisition of GmMAX2a Gene
PCR amplification was performed using pCAMBIA1300-GmMAX2a obtained in step 4 of example 1 as a template and primers Primer-ES and Primer-EAS to obtain a PCR amplification product, namely GmMAX2a gene. The primer sequences were as follows:
Primer-ES：5’-ACGATGATAAGGGCGGTACCAATATCTTACCGGAGAATGGGC-3’；
Primer-EAS：5’-AGGCTACGTAGGATCCTTACATGTCAATCACATATTCGACG-3’。
PCR amplification System (50 μl): cDNA 1. Mu.l, primer-F1. Mu.l, primer-R1. Mu.l, prime Star Mix 12.5. Mu.l, ddH 2 O 9.5μl。
PCR amplification conditions: 98℃10s,52℃10s,72℃2min40s,35 cycles; 72 ℃ for 5min; the reaction was terminated at 4 ℃.
2. Plant expression vector acquisition
And (3) carrying out enzyme digestion and connection on the pCAMBIA1300 vector and the PCR amplification product by using restriction enzyme to obtain a recombinant vector pCAMBIA1300-GmMAX2a, and carrying out sequencing verification on the recombinant vector pCAMBIA1300-GmMAX2 a.
Sequencing results showed that: the recombinant vector pCAMBIA1300-GmMAX2a is obtained by replacing a DNA fragment between enzyme cutting sites of the pCAMBIA1300 vector with a GmMAX2a gene shown in SEQ ID NO.3 and keeping other sequences of the pCAMBIA1300 vector unchanged.
3. Transformation
The recombinant vector pCAMBIA1300-GmMAX2a is transformed into agrobacterium tumefaciens GV3101 by adopting a freeze thawing method, and a positive transformant (a transformant containing the GmMAX2a gene shown in a sequence 1 in a sequence table) is obtained through PCR identification and is used for infecting an arabidopsis plant.
4. Acquisition of transgenic GmMAX2a Arabidopsis thaliana
Infecting wild Arabidopsis thaliana (Col-0) with Agrobacterium containing recombinant vector pCAMBIA1300-GmMAX2a by a Floral-dip method, and culturing the infected Arabidopsis thaliana to obtain T 0 Substitution of GmMAX2a arabidopsis seeds. Will T 0 After the surface of the seed of the arabidopsis thaliana transformed into GmMAX2a is disinfected, the seed is sown on a 1/2MS culture medium containing 25mg/L of fixed weed (glufosinate-ammonium, sigma, 45520) for screening to obtain T 1 And replacing GmMAX2a Arabidopsis seedlings. Repeating the steps until T is obtained 3 Substitution GmMAX2a arabidopsis homozygous line.
Extraction of T 3 And carrying out RT-PCR identification by replacing genomic DNA of the Arabidopsis thaliana seedlings of GmMAX2 a. The method comprises the following specific steps:
extracting T obtained in step one 3 Carrying out substitution conversion on total RNA of the Arabidopsis plant GmMAX2a, and carrying out reverse transcription to obtain cDNA; and (3) taking cDNA AS a template, respectively adopting Primer-qS and Primer-qAS Primer pairs, and an action S and an action AS Primer pair, and detecting the expression quantity of the GmMAX2a gene by RT-PCR to obtain a PCR amplification product. The primer sequences are shown below:
Primer-qS：5’-ACGATGATAAGGGCGGTACCAATATCTTACCGGAGAATGGGC-3’；
Primer-qAS：5’-AGGCTACGTAGGATCCTTACATGTCAATCACATATTCGACG-3’。
Actin S：5’-TTACCCGATGGGCAAGTC-3’；
Actin AS：5’-GCTCATACGGTCAGCGATAC-3’。
PCR amplification System [ (]50μl)：cDNA 1μl，Primer-F 1μl，Primer-R 1μl，Prime Star Mix 12.5μl， ddH 2 O 9.5μl。
PCR amplification conditions:
GmMAX2a: stopping the reaction at the temperature of [98 ℃ for 10 s-52 ℃ for 10 s-72 ℃ for 2min40s ]. Times.35-72 ℃ for 5 min-4 ℃;
action 2: [98 ℃ for 10 s- & gt 52 ℃ for 10 s- & gt 72 ℃ for 2min40s ]. Times.35- & gt 72 ℃ for 5 min- & gt 4 ℃ for terminating the reaction.
The PCR amplified products were subjected to 1% agarose gel electrophoresis, and the detection results are shown in FIG. 4: RT-PCR of wild type Arabidopsis plants does not have amplification products, whereas T 3 Substitution GmMAX2a arabidopsis homozygous line #5 and T 3 The generation-transferred GmMAX2a arabidopsis homozygous line #8 can amplify a target band, which indicates that the exogenous gene GmMAX2a gene is smoothly integrated on the genome of the arabidopsis, and can be normally transcribed and expressed in the transgenic arabidopsis. Selecting T 3 Substitution GmMAX2a Arabidopsis homozygous lines #5 and T 3 The substitution GmMAX2a arabidopsis homozygous line #8 was used for the next step of phenotyping.
2. Phenotypic analysis of GmMAX2 a-transformed Arabidopsis plants under salt and alkali stress
1. Seedling stage phenotype and root length of GmMAX2 a-transformed Arabidopsis thaliana under salt and alkali treatment
Selecting full wild Arabidopsis thaliana (Columbia ecotype), T 3 Substitution GmMAX2a Arabidopsis homozygous lines #5 and T 3 Seed of homozygous strain #8 of Arabidopsis with substitution GmMAX2a, sterilized with 5% sodium hypochlorite sterilizing solution for 10min, vernalized at 4deg.C for 3d, and sown on 1/2MS solid medium. After 5d, the arabidopsis thaliana and the wild arabidopsis thaliana seedlings which are transformed into GmMAX2a and have consistent growth vigor are selected and placed at the level of NaHCO with different concentrations of NaCl (75 mmol/L, 100mmol/L, 125 mmol/L) 3 (0.5 mmol/L, 0.6mmol/L, 0.7 mmol/L) stress medium, and after 7d the fresh weight (weighing once per 10 seedlings) and root length of Arabidopsis thaliana of each treatment group and non-treatment group were recorded for observation, and all experimental techniques were repeated and biological repeated 3 times each. Each strain was 10 strains per experiment.
The results are shown in fig. 5 and 6: under normal conditions (i.e. without any stressProcess, control), T 3 Substitution GmMAX2a Arabidopsis homozygous lines #5 and T 3 The generation-transformed GmMAX2a arabidopsis homozygous line #8 has no obvious difference with the growth and development of the wild type arabidopsis, which indicates that the introduced GmMAX2a gene does not influence the growth and development of the arabidopsis in the seedling stage.
And after 75mmol/L, 100mmol/L and 125mmol/L NaCl are added into the culture medium, the growth of the three strains is inhibited to different degrees, wherein the root length of the transgenic strains #5 and #8 is obviously longer than that of the WT when the 75mmol/L NaCl is stressed, the leaves are bigger and the color is darker, the WT shows cotyledon whitening phenomenon when the 125mmol/L NaCl is stressed, the growth is severely inhibited, the plants almost die, the growth of the transgenic strains is obviously inhibited, and the growth condition is overall better than that of the WT. The statistical results of root length and fresh weight show that the seedling main root length and fresh weight of transgenic lines #5 and #8 under salt stress are higher than those of WT, which indicates that GmMAX2a gene can participate in the response of plants to salt stress, so that the adaptability to salt stress is stronger.
At a concentration of 0.5mmol/L, 0.6mmol/L, 0.7mmol/L NaHCO 3 After stress in the culture medium, the growth vigor of the three strains is different and is 0.5mmol/L NaHCO 3 The difference of growth vigor in the culture medium is most obvious, 0.7mmol/L NaHCO 3 Root growth of WT and transgenic lines #5 and #8 in the culture medium is greatly affected, and the growth of the lateral roots is mainly restrained, and leaves are wilted. The statistical results of root length and fresh weight are similar to those of salt stress, and the main root length and fresh weight of seedlings of transgenic lines #5 and #8 are higher than those of WT, and the results show that the GmMAX2a gene can reduce the damage of alkali stress to the growth and development of transgenic arabidopsis seedlings, but along with NaHCO 3 The concentration is increased, the growth inhibition effect on each strain is enhanced, and the difference between root length and fresh weight of the WT and the two over-expressed strains is gradually reduced.
Example 4 determination of the expression level of transgenic Arabidopsis salt and alkali stress-related Marker Gene
1. Treatment of plant material
And cutting the obtained plant material GmMAX2a transgenic overexpression Arabidopsis thaliana strain #8 serving as a tissue sample to be tested, rapidly putting the tissue sample into liquid nitrogen for freezing, and then storing the tissue sample at the temperature of minus 80 ℃.
2. Extraction of Total RNA and cDNA acquisition
Extracting total RNA of the tissue samples to be tested after different time treatments obtained in the step 1 by adopting a Plant Total RNA Isolation Kit kit (Foregene); the cDNA is obtained by reverse transcription using the obtained total RNA as a template.
3、Real-time PCR
And (3) taking the cDNA as a template, adopting a Marker gene primer related to salt and alkali stress, and detecting the expression quantity of the GmMAX2a gene by Real-time PCR. The primer sequences are shown below:
salt stress related Marker genes: RD29B, SOS, NXH1, atRD22, KIN1, 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)；
Alkali stress related Marker genes: RD29A, COR, COR15A, KIN1, H+ -APase, 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)；
Conditions for Real-time PCR reactions: 94℃for 10 min- & gt [94℃for 30 s- & gt 64℃for 30s ]. Times.40- & gt 60℃for 30 s- & gt 72℃for 10 min- & gt 94℃for 2min.
Real-time PCR adopts comparative CT method ΔΔ CT) calculating gene expression amount, taking wild soybean GsGAPDH gene as reference gene and untreated sample as control. The target gene expression differences are expressed as fold-over of the treated samples relative to the untreated samples at each time point. Each sample included 3 biological replicates and 3 technical replicates, the data averaged 3 biological replicates, and if there was a large variance in one value, the two data averaged. The original data is normalized. The data after normalization were subjected to a difference significance analysis by T-test. The relative expression amount calculating method comprises the following steps: 2-ΔΔ CT＝ 2- ( Δ CT treatment Δ CT control) = 2- [ (CT treatment objective Gene-CT treatment reference Gene) - (CT control objective Gene-CT control reference Gene)]. The primer sequences of the internal reference genes are shown as follows:
Actin2-S：5’-TTACCCGATGGGCAAGTC-3’；
Actin2-AS：5’-GCTCATACGGTCAGCGATAC-3’。
the results are shown in fig. 7 and 8: under salt stress, the relative expression quantity of 6 Marker genes in GmMAX2a over-expressed transgenic Arabidopsis is higher than 0h between 3 and 6h, and the expression quantity in GmMAX2a over-expressed transgenic Arabidopsis is obviously higher than WT. Wherein, the relative expression level of RD29B, SOS1, NXH1, atRD22 and COR15A in GmMAX2a over-expressed transgenic Arabidopsis is higher than that of WT and the difference is very obvious when stress is 6 h; the expression quantity of KIN1 is extremely obviously different after 3 hours of salt stress.
Through 50mmol/L NaHCO 3 After 3h and 6h treatment, the relative expression amounts of the 6 alkali stress related Marker genes in the WT and GmMAX2a transgenic Arabidopsis are analyzed, and the relative expression amounts of the 6 Marker genes in the GmMAX2a transgenic Arabidopsis are all obviously higher than 0h. The relative expression amounts of RD29A, COR A, KIN1, H+ -APase and NADP-ME genes in the GmMAX2a transgenic Arabidopsis thaliana reach extremely significant levels when the alkali treatment is carried out for 3 hours, and the expression amounts also have a descending trend along with the prolonged treatment time, but are higher than that of the WT; the expression level of COR47 is always higher than that of WT, and the relative expression level of genes in alkaline treatment for 3h and 6h reaches an extremely remarkable level.
The results show that under salt and alkali conditions, the adverse stress marker genes such as RD29B, COR15A, RD A are up-regulated in GmMAX2a over-expressed transgenic Arabidopsis thaliana, and the expression quantity is obviously higher than that of WT, so that the GmMAX2a gene can enhance the tolerance to salt and alkali stress by regulating the expression level of the adverse stress induction marker genes.
Fresh weight data (g) under different stresses:
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 |
Alkali stress:
TABLE 2NaHCO 3 0.5mM
| 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 3NaHCO 3 0.6mM
TABLE 4NaHCO 3 0.7mM
| 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 6NaCl 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 7NaCl 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 (cm) under different stresses:
TABLE 11CK
Table 12
TABLE 13
TABLE 14
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TABLE 15
Table 16
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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.
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| CN103923888A (en) * | 2014-03-11 | 2014-07-16 | 中国农业科学院作物科学研究所 | Cotton GhMAX2b gene, coding protein and application |
| CN105585620A (en) * | 2016-03-07 | 2016-05-18 | 哈尔滨师范大学 | Soybean protein GmAIRP1 and application of coding gene of soybean protein GmAIRP1 in stress resistance plant cultivation |
| CN106749584A (en) * | 2017-01-19 | 2017-05-31 | 东北农业大学 | A kind of and plant alkali resistance GAP-associated protein GAP GsERF71 and its encoding gene and application |
| CN107058380A (en) * | 2016-11-03 | 2017-08-18 | 山东农业大学 | Application of the MdMAX2 genes in stress resistance of plant is improved |
-
2022
- 2022-07-10 CN CN202210819798.7A patent/CN115873085B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103923888A (en) * | 2014-03-11 | 2014-07-16 | 中国农业科学院作物科学研究所 | Cotton GhMAX2b gene, coding protein and application |
| CN105585620A (en) * | 2016-03-07 | 2016-05-18 | 哈尔滨师范大学 | Soybean protein GmAIRP1 and application of coding gene of soybean protein GmAIRP1 in stress resistance plant cultivation |
| CN107058380A (en) * | 2016-11-03 | 2017-08-18 | 山东农业大学 | Application of the MdMAX2 genes in stress resistance of plant is improved |
| CN106749584A (en) * | 2017-01-19 | 2017-05-31 | 东北农业大学 | A kind of and plant alkali resistance GAP-associated protein GAP GsERF71 and its encoding gene and application |
Non-Patent Citations (7)
| Title |
|---|
| AppleF-BoxProteinMd MAX2RegulatesPlantPhot omorphogenesisandStressR esponse;Jian-Ping An 等;Frontiers in Plant Science;第7卷;Article168 5 * |
| NAVEED UR REHMAN.大豆独脚金内酯合成和信号路径相关基因全基因组分析及几个GmMAX基因功能分析.《中国博士学位论文全文数据库农业科技辑》.2020,(2020年第05期),摘要第2段. * |
| SMXLs regulate seed germination under salinity and drought stress in soybean;Xujun Fu等;Plant Growth Regulation;第(2022) 96卷;397–408 * |
| XP_014632266.1.GenBank.2021,FEATURES和ORIGIN部分. * |
| Xujun Fu等.SMXLs regulate seed germination under salinity and drought stress in soybean.Plant Growth Regulation.2022,第(2022) 96卷397–408. * |
| 大豆独脚金内酯合成和信号路径相关基因全基因组分析及几个GmMAX 基因功能分析;NAVEED UR REHMAN;《中国博士学位论文全文数据库农业科技辑》;参见全文 * |
| 大豆独脚金内酯合成和信号路径相关基因全基因组分析及几个GmMAX基因功能分析;NAVEED UR REHMAN;《中国博士学位论文全文数据库农业科技辑》(2020年第05期);摘要第2段 * |
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