CN107383179B - A protein GsSLAH3 related to plant stress tolerance and its coding gene and application - Google Patents

Abstract

The invention discloses a kind of and plant stress tolerance correlative protein GsSLAH3 and its encoding gene and applications.The present invention is cloned from wild soybean obtains GsSLAH3 gene, carbonate suspension stress-inducing up-regulated expression；Tissue positioning analysis shows that GsSLAH3 gene is mainly expressed in the tissue such as root, stem, hypocotyl and fruit pod；By its transient expression in onion epidermis cell, discovery fusion protein fluorescence signal is mainly appeared in cell membrane；By GsSLAH3 gene overexpression in arabidopsis, discovery GsSLAH3 gene can enhance arabidopsis to the patience of carbonic acid salt stress.The present invention is to cultivate alkaline-resisting New Crop Varieties to have established Research foundation, to development and utilization saline alkali land resource important in inhibiting.

Description

A protein GsSLAH3 related to plant stress tolerance and its coding gene and application

technical field

The invention belongs to the field of biotechnology, and specifically relates to a protein GsSLAH3 related to plant stress tolerance, its coding gene and application.

Background technique

The area of saline-alkali land in my country is as high as 1.5 billion mu, and there are as many as 55 million mu in the northeast region alone. Saline-alkali adverse environment seriously harms the growth and development of crops, and is a major problem restricting agricultural production in our country. Heilongjiang Province has tens of millions of mu of salinized land. The development and utilization of these land resources has important practical and strategic significance for increasing my country's agricultural output and ensuring food security, and will also create huge ecological and social benefits. The saline-alkali land in our province is mainly alkaline soil containing Na ₂ CO ₃ and NaHCO ₃ . Compared with neutral salts such as NaCl and Na ₂ SO ₄ , carbonate not only causes osmotic stress and ion poisoning, but also increases the High pH damage caused by CO ₃ ^2- and HCO ₃ ^- etc., resulting in mixed toxic effects. Therefore, the mechanism of carbonate stress is more complicated than that of neutral salt, and the damage to plants is greater.

Improving the salt-alkali tolerance of crops has become a hot and difficult point in the current technical research in the field of agriculture and animal husbandry, and it is also a major problem that needs to be solved urgently. In recent years, with the development of functional genomics and molecular biology, excavating the key genes of salt-alkali tolerance and using genetic engineering technology to cultivate new crop varieties with good salt-alkali tolerance have become one of the means to effectively improve the salt-alkali tolerance of crops. one. Wild soybean can survive in low temperature and highly salinized land, and has the characteristics of wide adaptability and strong stress tolerance. It is an ideal donor material for obtaining saline-alkali tolerance genes.

Anion channels are widely involved in physiological processes such as plant organic acid secretion, membrane potential change, turgor control, and crop salt tolerance. Combined with studies in model plants Arabidopsis and rice, the slow-type anion channel (S-type anion channel) plays an important role in plant responses to abiotic stresses such as light, drought, and salt. Therefore, excavating wild soybean slow-type anion channel protein genes will provide functionally significant genetic resources for stress-tolerant molecular breeding of crops.

Contents of the invention

The technical problem to be solved by the invention is how to regulate the stress tolerance of plants.

To solve the above technical problems, the present invention firstly provides a protein related to plant stress tolerance.

The name of the protein related to plant stress tolerance provided by the present invention is GsSLAH3, which is the protein of a) or b) or c) or d) as follows:

a) the amino acid sequence is the protein shown in Sequence 2;

b) a fusion protein obtained by connecting a tag to the N-terminal and/or C-terminal of the protein shown in Sequence 2;

c) a protein having the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in Sequence 2;

d) A protein having 75% or more homology with the amino acid sequence shown in Sequence 2 and having the same function.

Among them, sequence 2 consists of 561 amino acid residues.

In order to make the protein in a) easy to purify, the amino terminus or carboxyl terminus of the protein shown in Sequence 2 in the Sequence Listing can be linked with the tags shown in Table 1.

Table 1. Sequence of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

For the protein GsSLAH3 in c) above, the substitution and/or deletion and/or addition of one or several amino acid residues is a substitution and/or deletion and/or addition of no more than 10 amino acid residues.

The protein GsSLAH3 in the above c) can be synthesized artificially, or its coding gene can be synthesized first, and then biologically expressed.

The gene encoding the protein GsSLAH3 in the above c) can be obtained by deleting the codon of one or several amino acid residues in the DNA sequence shown in sequence 1, and/or performing a missense mutation of one or several base pairs, and /or obtained by linking the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end.

In order to solve the above technical problems, the present invention further provides biological materials related to GsSLAH3 protein.

The biological material related to the GsSLAH3 protein provided by the present invention is any one of the following A1) to A12):

A1) a nucleic acid molecule encoding a GsSLAH3 protein;

A2) an expression cassette containing the nucleic acid molecule of A1);

A3) a recombinant vector containing the nucleic acid molecule of A1);

A4) a recombinant vector containing the expression cassette described in A2);

A5) a recombinant microorganism containing the nucleic acid molecule of A1);

A6) a recombinant microorganism containing the expression cassette described in A2);

A7) A recombinant microorganism containing the recombinant vector described in A3);

A8) a recombinant microorganism containing the recombinant vector described in A4);

A9) a transgenic plant cell line containing the nucleic acid molecule of A1);

A10) a transgenic plant cell line containing the expression cassette described in A2);

A11) a transgenic plant cell line containing the recombinant vector described in A3);

A12) A transgenic plant cell line containing the recombinant vector described in A4).

In the above-mentioned biological material, the nucleic acid molecule described in A1) is the gene shown in 1) or 2) or 3) as follows:

1) its coding sequence is a cDNA molecule or a genomic DNA molecule shown in Sequence 1;

2) A cDNA molecule or a genomic DNA molecule that has 75% or more identity to the nucleotide sequence defined in 1) and encodes the GsSLAH3 protein;

3) A cDNA molecule or a genomic DNA molecule that hybridizes to the nucleotide sequence defined in 1) or 2) under stringent conditions and encodes the GsSLAH3 protein.

Wherein, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

Among them, Sequence 1 consists of 1686 nucleotides, encoding the amino acid sequence shown in Sequence 2.

Those skilled in the art can easily use known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence encoding GsSLAH3 of the present invention. Those artificially modified nucleotides with 75% or higher identity to the nucleotide sequence of GsSLAH3 isolated in the present invention, as long as they encode GsSLAH3 and have the same function, are all derived from the nucleotide sequence of the present invention And is equivalent to the sequence of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher, of the nucleotide sequence of the protein composed of the amino acid sequence shown in the coding sequence 2 of the present invention. Nucleotide sequences of higher identity. Identity can be assessed visually or with computer software. Using computer software, identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

The identity of 75% or more may be 80%, 85%, 90% or more.

Among the above-mentioned biological materials, the expression cassette (GsSLAH3 gene expression cassette) described in A2) that contains the nucleic acid molecule encoding GsSLAH3 refers to the DNA that can express GsSLAH3 in the host cell, and the DNA can not only include a promoter that initiates GsSLAH3 transcription, A terminator that terminates transcription of GsSLAH3 may also be included. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to: constitutive promoters; tissue, organ and development specific promoters and inducible promoters. Suitable transcription terminators include, but are not limited to: Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine Synthase terminator.

The existing expression vector can be used to construct the recombinant vector containing the expression cassette of the GsSLAH3 gene. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Company), etc. The plant expression vector may also include the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopaline synthase gene Nos), plant gene (such as soybean The untranslated region transcribed at the 3′ end of the storage protein gene) has similar functions. When using the gene of the present invention to construct plant expression vectors, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be ATG initiation codons or adjacent region initiation codons, etc. The reading frames of the sequences are identical to ensure correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes (GUS gene, luciferase gene, etc.) genes, etc.), antibiotic marker genes (such as the nptII gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinothricin, and the hph gene that confers resistance to the antibiotic hygromycin , and the dhfr gene that confers resistance to methotrexate, the EPSPS gene that confers resistance to glyphosate) or the chemical resistance marker gene (such as the herbicide resistance gene), the mannose-6- that provides the ability to metabolize mannose Phosphate isomerase gene. Considering the safety of the transgenic plants, the transformed plants can be screened directly by adversity without adding any selectable marker gene.

In the above biological materials, the vector can be a plasmid, a cosmid, a phage or a viral vector.

In the above biological materials, the microorganisms can be yeast, bacteria, algae or fungi, such as Agrobacterium.

Among the above biological materials, the transgenic plant cell lines do not include propagation materials.

In order to solve the above-mentioned technical problems, the present invention also provides new applications of the GsSLAH3 protein or the above-mentioned biological materials.

The invention provides the application of the GsSLAH3 protein or the above-mentioned biological material in regulating the stress tolerance of plants.

In the above-mentioned application, the regulation is to improve.

The present invention also provides the application of the GsSLAH3 protein or the above-mentioned biological material in cultivating transgenic plants with improved stress tolerance.

The present invention also provides the application of the GsSLAH3 protein or the above-mentioned biological material in plant breeding.

In the above application, the stress resistance is alkali resistance; the alkali resistance is specifically resistance to carbonate stress; and the resistance to carbonate stress is specifically resistance to NaHCO ₃ stress.

In the above-mentioned application, the plant is a monocotyledon or a dicotyledon, and the dicotyledon can specifically be a leguminous plant and/or a cruciferous plant and/or a compositae plant; the described leguminous plant can be a soybean, Lotus japonicus, alfalfa or pumice; the cruciferous plant can be Arabidopsis thaliana or rapeseed; the composite family plant can be sunflower; the Arabidopsis can be Arabidopsis thaliana (Columbian ecotype col-0 ).

In order to solve the above technical problems, the present invention finally provides a method for cultivating transgenic plants with improved stress tolerance.

The method for cultivating transgenic plants with improved stress tolerance provided by the present invention includes the step of increasing the expression level and/or activity of GsSLAH3 protein in recipient plants to obtain transgenic plants; the stress tolerance of the transgenic plants is higher than that of the recipient plant.

In the above method, the method for increasing the expression level and/or activity of the GsSLAH3 protein in the recipient plant is to overexpress the GsSLAH3 protein in the recipient plant.

In the above method, the overexpression method is to introduce the gene encoding the GsSLAH3 protein into the recipient plant; the nucleotide sequence of the gene encoding the GsSLAH3 protein is the DNA molecule shown in Sequence 1.

In one embodiment of the present invention, the gene encoding the GsSLAH3 protein (ie, the nucleotide shown in Sequence 1) is introduced into Agrobacterium LBA4404 through the recombinant vector pCAMBIA230035S-GsSLAH3 containing the expression cassette of the gene encoding the GsSLAH3 protein. The recombinant vector pCAMBIA230035S-GsSLAH3 is a vector obtained by replacing the DNA fragment between the SmaI and XbaI restriction sites of the pCAMBIA230035S vector with the GsSLAH3 gene shown in sequence 1 in the sequence listing, and keeping other sequences of the pCAMBIA230035S vector unchanged . The recombinant vector pCAMBIA230035S-GsSLAH3 expresses GsSLAH3 protein.

In the above method, the stress resistance is alkali resistance; specifically, the alkali resistance is resistance to carbonate stress.

In the above method, the tolerance to carbonate stress is resistance to NaHCO ₃ stress, which is specifically embodied in the condition of NaHCO ₃ stress: the seed germination rate of the transgenic plant is higher than that of the recipient plant and/or the root length of the transgenic plant is longer than that of the recipient Plants and/or transgenic plants have higher aerial parts biomass than recipient plants and/or transgenic plants have higher chlorophyll content than recipient plants and/or transgenic plants have lower electrolyte extravasation than recipient plants. The NaHCO ₃ concentration may specifically be 4mM, 5mM, 7mM, 8mM and 150mM.

In the above method, the transgenic plant is understood to include not only the first-generation transgenic plant obtained by transforming the GsSLAH3 gene into a recipient plant, but also its progeny. For transgenic plants, the gene can be propagated in that species, or transferred into other varieties of the same species, particularly including commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

In the above method, the recipient plant is a monocotyledon or a dicotyledon, and the dicotyledon can specifically be a leguminous plant and/or a cruciferous plant and/or a compositae plant; the said leguminous plant can be Soybean, lotus root, alfalfa or pumice; The cruciferous plant can be Arabidopsis thaliana or rapeseed; The Compositae plant can be sunflower; The Arabidopsis thaliana can be Arabidopsis thaliana (Columbian ecotype col -0).

The present invention clones the GsSLAH3 gene from wild soybeans, and its expression is up-regulated by carbonate stress; tissue localization analysis shows that the GsSLAH3 gene is mainly expressed in tissues such as roots, stems, hypocotyls and fruit pods; it is transiently expressed in onions In epidermal cells, it was found that the fusion protein fluorescence signal mainly appeared in the cell membrane; GsSLAH3 gene was overexpressed in Arabidopsis, and it was found that GsSLAH3 gene could enhance the tolerance of Arabidopsis to carbonate stress. The invention lays a research foundation for cultivating new varieties of alkali-resistant crops, and has great significance for the development and utilization of saline-alkali land resources.

Description of drawings

Figure 1 is the expression pattern of GsSLAH3 gene in wild soybean roots treated with 50mM NaHCO ₃ .

Fig. 2 is the relative expression level of GsSLAH3 gene in different tissues of wild soybean.

Figure 3 is the subcellular localization analysis of GsSLAH3.

Figure 4 is the RT-PCR identification of transgenic GsSLAH3 Arabidopsis.

Figure 5 is a statistical analysis of the germination phenotype and germination rate of GsSLAH3 transgenic Arabidopsis plants treated with 7mM NaHCO ₃ and 8mM NaHCO ₃ .

Fig. 6 is a statistical analysis of the seedling phenotype, root length and aerial part biomass of GsSLAH3 transgenic Arabidopsis plants treated with 4mM NaHCO ₃ and 5mM NaHCO ₃ .

Fig. 7 is a statistical analysis of the seedling phenotype, chlorophyll content and electrolyte extravasation rate of GsSLAH3 transgenic Arabidopsis plants treated with 150 mM NaHCO ₃ .

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The quantitative tests in the following examples were all set up to repeat the experiments three times, and the results were averaged.

The wild-type soybean G07256 in the following examples is recorded in the following documents: Mingzhe Sun, Xiaoli Sun, Yang Zhao, Hua Cai, Chaoyue Zhao, Wei Ji, Huizi DuanMu, Yang Yu, YanmingZhu.Ectopic expression of GsPPCK3and SCMRP in Medicago sativa enhances plantalkaline stress tolerance and methionine content. PLOS ONE 2014,9(2):e89578, the public can obtain from the applicant (Heilongjiang Bayi Agricultural Reclamation University), this biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes use.

The pBSK-35S-eGFP vector in the following examples is described in the following documents: Xiaoli Sun, Wei Ji, Xiaodong Ding, Xi Bai, Hua Cai, Shanshan Yang, Xue Qian, Mingzhe Sun, YanmingZhu.GsVAMP72, a novel Glycine soja R -SNARE protein, is involved in regulating plant salt tolerance and ABA sensitivity.Plant Cell Tiss Organ Cult 2013,113:199–215, the public can obtain from the applicant (Heilongjiang Bayi Agricultural Reclamation University), the biological material is only for repeating the present invention It is used for related experiments and cannot be used for other purposes.

The pCAMBIA230035S vector in the following examples is described in the following documents: Ailin Liu, Yang Yu, Xiangbo Duan, Xiaoli Sun, Huizi Duanmu, Yanming Zhu. GsSKP21, a Glycine sojaSphase kinase associated protein, mediates the regulation of plant alkaline tolerance and ABA sensitivity. Plant Mol Biol (2015) 87:111–124 is available to the public from the applicant (Heilongjiang Bayi Agricultural Reclamation University). This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

Agrobacterium LBA4404 in the following examples is described in the following documents: Ailin Liu, Yang Yu, XiangboDuan, Xiaoli Sun, Huizi Duanmu, Yanming Zhu. GsSKP21, a Glycine soja Sphase kinase associated protein, mediates the regulation of plant alkaline tolerance and ABA sensitivity. Plant Mol Biol (2015) 87:111–124 is available to the public from the applicant (Heilongjiang Bayi Agricultural Reclamation University). This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

Embodiment 1, the cloning of wild soybean GsSLAH3 gene

1. Select the plump wild soybean G07256 seeds and treat them in concentrated sulfuric acid for 10 minutes to remove the mud film. After pouring out the concentrated sulfuric acid, wash them with sterile water for 3-4 times, place them on wet filter paper, and culture them in dark at 25°C for 3 days to accelerate germination. When the buds grow to about 1-2 cm, they are transferred to a bowl filled with Hoagland's culture medium, fixed with space cotton, immersed in the culture medium, and placed in an artificial climate box for cultivation.

2. When the seedlings grow to 3 weeks old, take 3 cm of the roots and put them into EP tubes, and store them at -80°C. The RNA was extracted according to the instructions of the RNAprep pure kit (TRANSGEN BIOTECH), and then the cDNA was synthesized.

3. Using the synthesized cDNA as a template, PCR amplification was performed with primers Primer-KS and Primer-KAS. The primer sequences are as follows:

Primer-KS: 5’–TATAACCTGTGCTTCTGATAGTGTGT–3’;

Primer-KAS: 5’–TGTTGGTTGCCTCATTATTTTCTT–3’.

PCR amplification system (50 μL): cDNA 4 μL, 10×PS buffer (Mg ²⁺ ) 10 μL, dNTP Mixture (2.5mM) 4 μL, Primer-KS 1 μL, Primer-KAS 1 μL, Prime Star DNA Polymerase (TaKaRa) 0.5 μL, ddH ₂ O 29.5 μL. The PCR amplification conditions were: 98°C for 8 min; 98°C for 10 s, 58°C for 10 s, 72°C for 2 min, 30 cycles; 72°C for 10 min; 4°C for termination.

The PCR amplified product was detected by 1.5% agarose gel electrophoresis to obtain a band with a molecular weight of about 1.9Kb, and the agarose gel recovery kit (TRANSGEN BIOTECH) was used to recover the PCR amplified product; it was combined with the pEASY-BluntSimple vector (TRANSGEN BIOTECH) to obtain the recombinant plasmid pEASY-Blunt Simple-GsSLAH3. And it was transformed into Escherichia coli DH5α competent cells and sent for sequencing.

Sequencing results show that: PCR amplification obtains an amplified product with a size of 1910bp, which contains a complete open reading frame (ORF) with a size of 1686bp, and the nucleotide sequence of a DNA fragment with a size of 1686bp is shown in sequence 1 in the sequence listing , and the gene shown in sequence 1 was named GsSLAH3 gene, the amino acid sequence encoded by the GsSLAH3 gene was shown in sequence 2 in the sequence listing, and the amino acid sequence shown in sequence 2 was named GsSLAH3 protein.

Embodiment 2, the expression characteristic analysis of GsSLAH3 gene

1. Analysis of the expression pattern of GsSLAH3 gene in wild soybean roots under alkaline stress

1. Select the plump wild soybean G07256 seeds and treat them in concentrated sulfuric acid for 10 minutes to remove the mud film. After pouring out the concentrated sulfuric acid, wash them with sterile water 3-4 times, place them on wet filter paper, and culture them in dark at 25°C for 3 days to accelerate germination. Remove it when it is about 1-2cm long, and use Hoagland's liquid medium for hydroponics.

2. When the wild soybean seedlings grow to 3 weeks old, they are treated under the condition of 50mM NaHCO ₃ (pH8.5) for 0h, 1h, 3h, 6h, 9h, 12h, 24h, and then quickly cut the young roots and place them at -80℃ save. Total RNA was extracted and reverse transcribed into cDNA as a template for later use.

3. Using the Real time-PCR method, the wild soybean GAPDH gene was used as an internal reference gene, and the untreated sample was used as a control to analyze the relative expression of GsSLAH3 gene at different time points after treatment. The relative expression of genes was calculated by the comparative _CT method (ΔΔCT): 2 _-ΔΔCT = 2 - ( ^{ΔCT treatment - ΔCT control)} = 2 ^{- [(CT treatment - CT internal reference) - (CT control - CT internal reference)]} . GsGAPDH and GsSLAH3 gene primers are as follows:

GsGAPDH-S: 5’–GACTGGTATGGCATTCCGTGT–3’;

GsGAPDH-AS: 5’–GCCCTCTGATTCCCTCCTTGA–3’;

GsSLAH3-S: 5'-AGAGCCAGCAAGCGGTGTC-3';

GsSLAH3-AS: 5'-GTAACTGTGGACCTCTCCAATGTT-3'.

PCR reaction conditions: 95°C for 2min→[95°C for 15s→60°C for 30s]×40→95°C for 1min→55°C for 1min→95°C for 30s.

The result is shown in Figure 1. It can be seen from the figure that under the treatment of 50mM NaHCO ₃ , the expression of GsSLAH3 gene was up-regulated and reached the highest value at 6h, and then the expression decreased relatively but remained relatively high compared to 0h. It shows that GsSLAH3 gene is an alkali stress response gene.

2. Analysis of tissue expression pattern of GsSLAH3 gene

1. Select the plump wild soybean G07256 seeds and treat them in concentrated sulfuric acid for 10 minutes to remove the mud film. After pouring out the net concentration, rinse with sterile water 3-4 times, place them on moist filter paper, and culture them in dark at 25°C for 3 days to accelerate germination. When it is about 1-2cm, it is transferred to a seedling pot filled with 30% peat soil and 70% common soil, and then placed in an artificial climate box for cultivation.

2. Different tissues of wild soybean (root, stem, young leaf, mature leaf, hypocotyl, seed embryo, fruit pod and flower) were taken and stored at -80°C respectively.

3. Extract RNA from different tissues of wild soybean and reverse transcribe it into cDNA as a template for future use.

4. According to the method in step 3 of step 1, the relative expression of GsSLAH3 gene in different tissues of wild soybean was detected.

The result is shown in Figure 2. It can be seen from the figure that the GsSLAH3 gene is mainly expressed in roots, stems, hypocotyls and fruit pods, and the expression level in the hypocotyl is the highest; while the expression level of GsSLAH3 gene in flowers, leaves and embryos is relatively low.

Example 3, analysis of subcellular localization of GsSLAH3 protein mediated by particle gun bombardment

1. Using the cDNA of wild soybean G07256 as a template, GsSLAH3-YS and GsSLAH3-YAS were used for PCR amplification to obtain the GsSLAH3 gene. The primer sequence is as follows (the sequence of the restriction site introduced is underlined, and the left side is the protective base):

GsSLAH3-YS: 5’–GGGTCGACATGGAAAACAAACCTTAAC–3’;

GsSLAH3-YAS: 5'–CG TCTAGA TGGCTCCCAAGTCTTAAATG–3'.

PCR amplification system: 20 μL 5×PrimeSTAR ^TM HS PCR buffer, 8 μL dNTP mix (A, G, T, C, each 2.5 mM), 2 μL upstream and downstream primers (10 μM), 1 μL diluted 100 times (plasmid containing the target gene ), 1 μL high-fidelity enzyme [PrimeSTAR DNA Polymerase (TaKaRa)], sterile ddH ₂ O make up volume (total volume 100 μL).

PCR reaction conditions: 98°C for 8 min; 98°C for 10 s, 60°C for 10 s, 72°C for 1.5 min, 30 cycles; 72°C for 10 min; 4°C to terminate the reaction.

2. Using restriction enzymes SalI and XbaI to perform double enzyme digestion on the above-mentioned PCR amplification product and the vector pBSK-35S-eGFP, and ligate to obtain a recombinant plasmid. The ligation reaction system was: 1 μL of 10×Buffer, 1 μL of T ₄ DNA ligase, the concentration ratio of the large vector fragment and the target gene was 1:1, the volume was supplemented with ddH ₂ O (total volume 10 μL), ligated overnight at 16°C.

3. The above-mentioned recombinant plasmid and the empty vector (pBSK-35S-eGFP) were bombarded into onion epidermal cells by the gene gun method (see the instruction manual of Bio-Rad Helios gene gun system in the United States for the specific method). After culturing in dark for 12 hours, the bombarded onion epidermal cells were cut out, mounted on slices, and the green fluorescence was observed under a confocal laser microscope. After the fluorescence of the recombinant protein was observed, the mounted slide was treated with 30% (w/v) sucrose solution to separate the plasmodium, and the fluorescence was further observed.

The result is shown in Figure 3. The fluorescent signal of the empty vector was expressed in the whole cell; while the green fluorescent signal of the recombinant plasmid was mainly concentrated on the cell membrane, and after plasmolysis, the fluorescent signal on the membrane was enhanced. It shows that GsSLAH3 mainly functions as a membrane protein.

Example 4, Obtaining of Transgenic GsSLAH3 Arabidopsis Plants and Analysis of Its Resistance to Carbonate Stress

1. Obtaining and molecular identification of transgenic GsSLAH3 Arabidopsis

1. Using the recombinant plasmid pEASY-Blunt Simple-GsSLAH3 in Example 1 as a template, PCR amplification was performed using GsSLAH3-ES and GsSLAH3-EAS primers to obtain the CDS region of the GsSLAH3 gene. The primer sequences are as follows:

GsSLAH3-ES: 5'–AGG CCCGGG ATGGAAAACAACCTTAACCGT–3';

GsSLAH3-EAS: 5'–TCG TCTAGA TCATGGCTCCCAAGTCTTAAATGG–3'.

PCR amplification system (20 μL): template 0.2 μL, 10× buffer 2 μL, dNTP Mixture 2 μL, Mg ²⁺ 1 μL, GsSLAH3-ES 0.6 μL, GsSLAH3-EAS 0.6 μL, KOD DNA Polymerase (TaKaRa) 0.4 μL, ddH ₂ O 13.2 μL.

PCR amplification conditions: 95°C for 2min; 95°C for 30s, 60°C for 30s, 72°C for 1.5min, 30 cycles; 72°C for 10min; 4°C to terminate the reaction.

2. Carry out double digestion and ligation of the pCAMBIA230035S vector and the PCR amplification product obtained in step 1 with restriction endonucleases SmaI and XbaI to obtain the recombinant plasmid pCAMBIA230035S-GsSLAH3. and validated by sequencing.

The sequencing results show that the recombinant vector pCAMBIA230035S-GsSLAH3 is obtained by replacing the DNA fragment between the SmaI and XbaI restriction sites of the pCAMBIA230035S vector with the GsSLAH3 gene shown in sequence 1 in the sequence table, and keeping other sequences of the pCAMBIA230035S vector unchanged Carrier. The recombinant vector pCAMBIA230035S-GsSLAH3 expresses the GsSLAH3 protein shown in Sequence 2.

3. The recombinant vector pCAMBIA230035S-GsSLAH3 was transformed into Agrobacterium tumefaciens LBA4404 by freeze-thaw method, and positive transformants were obtained by PCR identification, which were used to infect Arabidopsis plants.

4. Infect wild-type Arabidopsis thaliana (Columbian ecotype col-0) with Agrobacterium containing the recombinant plasmid pCAMBIA230035S-GsSLAH3 by dipping flowers. T ₁ generation seeds were harvested and selected on 1/2 MS medium containing 25 mg/L kanamycin (kana). The next generation produced from the seedlings obtained from the screening was then subjected to kana screening, and so on, and so on, and finally the homozygous line of T ₃ generation transgenic GsSLAH3 Arabidopsis was obtained.

5. Extract the total RNA of the Arabidopsis thaliana transgenic GsSLAH3 plants of the T _3rd generation, and qualitatively detect the relative expression of the GsSLAH3 gene by RT-PCR. The sequences of the gene primers are the same as those in Example 2. At the same time, Actin2 was used as an internal reference gene, and the primer sequences were as follows:

Actin2-S: 5'-TTACCCGATGGGCAAGTC-3';

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

PCR reaction system: 5 μL 2×Easy Taq DNA Polymerase, 0.8 μL upstream primer (10 μM), 0.8 μL downstream primer (10 μM), 1 μL cDNA diluted 100 times, sterile ddH ₂ O to make up volume (total volume 10 μL).

PCR amplification conditions: GsSLAH3: 94°C for 10min→[94°C for 30s→60°C for 30s→72°C for 90s]×30→72°C for 10min→4°C to terminate the reaction;

Actin2: 94°C for 10min→[94°C for 30s→60°C for 30s→72°C for 90s]×28→72°C for 10min→4°C to terminate the reaction.

The PCR products were detected by agarose gel electrophoresis, and some results are shown in Figure 4. It can be seen from the figure that the RT-PCR of wild-type Arabidopsis plants has no amplified products, while the homozygous lines #14 and #15 of Arabidopsis thaliana transfected with GsSLAH3 in the T ₃ generation can amplify the target bands, indicating that The exogenous gene GsSLAH3 has not only successfully integrated into the Arabidopsis genome, but also can be normally transcribed and expressed in the transgenic Arabidopsis. Homozygous lines #14 and #15 of T ₃ transgenic GsSLAH3 Arabidopsis lines were selected for phenotypic analysis in the next step.

2. Phenotype analysis of transgenic GsSLAH3 Arabidopsis under alkali stress

1. The germination phenotype and germination rate of Arabidopsis transgenic GsSLAH3 under alkali treatment

Select plump seeds of wild-type Arabidopsis thaliana and Arabidopsis homozygous lines #14 and #15 transduced with _GsSLAH3 in the T3 generation, disinfect with 5% sodium hypochlorite for 5 minutes, and then rinse with sterilized ddH ₂ O for 3-5 times. Placed at 4°C for 3 days. Then the seeds were sowed on 1/2MS medium containing 0mM, 7mM and 8mM NaHCO ₃ , cultured at 22°C for 3 days, and the phenotype was observed every day and the germination rate of the seeds was counted. The experiment was repeated three times, and 30 plants were used for each line in each treatment.

The result is shown in Figure 5. It can be seen from the figure that under normal conditions (the control group treated with 0mM NaHCO ₃ ), there was no significant difference in the germination status and germination rate of wild-type Arabidopsis and GsSLAH3-transformed Arabidopsis, indicating that GsSLAH3 has no effect on Arabidopsis. However, under the treatment of 7mM and 8mM NaHCO ₃ , the germination of wild-type Arabidopsis and GsSLAH3-transformed Arabidopsis were inhibited to a certain extent, but compared with wild-type Arabidopsis, GsSLAH3-transformed Arabidopsis The germination rate of the seeds was higher, which was significantly higher than that of the wild type. Therefore, overexpression of GsSLAH3 gene increased the tolerance of Arabidopsis to alkaline stress during germination.

2. Seedling phenotype, root length and fresh weight of transgenic GsSLAH3 Arabidopsis under alkali treatment

Select plump seeds of wild-type Arabidopsis thaliana and Arabidopsis homozygous lines #14 and #15 transduced with _GsSLAH3 in the T3 generation, disinfect with 5% sodium hypochlorite for 5 minutes, and then rinse with sterilized ddH ₂ O for 3-5 times. Laminate at 4°C for 3 days. The seeds were then sown on 1/2 MS solid medium. After 1 week, the wild-type Arabidopsis and GsSLAH3-transformed Arabidopsis seedlings with the same growth were selected and transferred to the medium containing 0mM, 4mM and 5mM NaHCO ₃ for vertical culture, and the Arabidopsis in the alkali stress treatment group and the control group were observed. Mustard growth, after 7 days, the root length and aboveground biomass (fresh weight) were counted.

The result is shown in Figure 6. It can be seen from the figure that under normal conditions (the control group treated with 0mM NaHCO ₃ ), there was no significant difference in the growth state, root length and fresh weight of wild-type Arabidopsis and transgenic GsSLAH3 Arabidopsis #14, #15 ; while under the treatment of 4mM and 5mM NaHCO ₃ , the growth of wild-type Arabidopsis and GsSLAH3-transformed Arabidopsis were all inhibited, but the inhibition degree of wild-type Arabidopsis was more severe than that of GsSLAH3-transformed Arabidopsis: Trans-GsSLAH3 Arabidopsis The root length and above-ground biomass of A. thaliana were significantly higher than those of the wild type. Therefore, overexpression of GsSLAH3 gene enhanced the tolerance of Arabidopsis to alkaline stress in seedling stage.

3. Determination of seedling phenotype and physiological indicators of transgenic GsSLAH3 Arabidopsis under alkali treatment

The plump seeds of wild-type Arabidopsis thaliana and _GsSLAH3 homozygous lines #14 and #15 of the T3 generation transgenic Arabidopsis thaliana were selected, stratified at 4°C for 3 days, and then sown in nutrient pots (nutrient soil, clivia soil, leech Stone mixed according to 1:1:1), placed in the greenhouse for cultivation (22 ℃, light 16h/d). After 3 weeks, the seedlings in the alkali treatment group were irrigated with 150mM NaHCO ₃ solution every 3-4 days. 20d observation (alkali) treatment group and the Arabidopsis growth of untreated group, and measure the chlorophyll content and electrolyte extravasation rate of treatment group and untreated group (detection method sees literature "Plant Physiological Experiment/Hao Zaibin etc. edited by Harbin Institute of Technology Publishing Society, 2004.9").

The result is shown in Figure 7. It can be seen from the figure that under the treatment of 150mM NaHCO ₃ , the leaves of wild-type Arabidopsis thaliana showed yellowing, curling, and wilting; while the chlorosis of Arabidopsis transgenic GsSLAH3 was relatively less, and the leaves were only slightly yellowing. The results of chlorophyll content measurement showed that after 150mM NaHCO ₃ treatment, although the chlorophyll content of wild-type Arabidopsis and GsSLAH3-transformed Arabidopsis both decreased, the reduction range of GsSLAH3-transformed Arabidopsis was significantly lower than that of wild-type Arabidopsis, indicating that wild-type Arabidopsis The photosynthetic system of the plant has been more damaged. Abiotic stress usually leads to plant electrolyte extravasation, so the rate of electrolyte extravasation can reflect the degree of plant damage. After 150mM NaHCO ₃ treatment, the electrolyte extravasation rates of wild-type Arabidopsis and GsSLAH3-transformed Arabidopsis increased, indicating that all lines were damaged to varying degrees, but compared with wild-type Arabidopsis, the electrolyte extravasation rate of transgenic Arabidopsis The electrolyte extravasation rate of GsSLAH3 Arabidopsis was significantly lower than that of the wild type, indicating that it was relatively less damaged by alkali stress. Therefore, the overexpression of GsSLAH3 gene improved the tolerance of Arabidopsis to alkali stress at the seedling stage.

The above results indicated that the overexpression of GsSLAH3 gene significantly improved the stress tolerance of plants, especially the alkali tolerance, and the GsSLAH3 gene could positively regulate the alkali tolerance of Arabidopsis.

sequence listing

<110> Heilongjiang Bayi Agricultural University

<120>A protein GsSLAH3 related to plant stress tolerance and its coding gene and application

<160>2

<210>1

<211>1686bp

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>1

atggaaaaca accttaaccg tgaaacagaa gggcatggct tgcctaaaat tccatcacta 60

gttcaacata tatcatcaaa tgatgaagga aactttgaca atggtgactt gaaaaatcta 120

agctcatctt tcaaagagaa tgaaacaatc ttagcaggaa gccaaggtga tgaacatgca 180

gccattaatc atcggaataa gcattcggta tctatcaaca taccattctc ttgtgaagag 240

gttcagctgc ataacaccgt aagagttctc tatagtggcg aaaatgattt ctcttctcaa 300

tcaactacta cagactccaa gccaccatta ccatcacaat caatgccaaa tggcagtctg 360

cactcagagc cagcaagcgg tgtcaatttt aacaatcaag aaagcgtcat aagcttgaat 420

aataagagga ttgatttttt caaaacatgg tccagtaaac taggggggaca catatcagtt 480

atgagtggaa aggtacatac agaaagtgca gaagatgata acagcttatg caacactaat 540

aagcctttac ctgttgattt gttcttcaaa acattggagg gtccacagtt acaaactcct 600

aagaagtctt cagaagagat ggtgcttcct caggacaagc agtggccatt tcttcttcgg 660

tttccggttt catcttttgg tatttgtctt ggagttagca gtcaagcaat tctttggaaa 720

gcattggcca cgtctccttc cactgcattt cttcacataa cccctaaaat aaatttcatc 780

ctgtggttca tctccattgg tattgttgct actattttca ccacctatct cttcaaaata 840

attctccatt ttgaagcagt tcgtcgtgag taccaacatc cggttcgtgt taacttcttc 900

tttgcaccat ggatagccct tttgttccta gctcttggag ttcccccatc agttaccaag 960

gacttgcatc aagcagtttg gtacattcta atgattccat tgttctgcct taagctaaag 1020

atatatggac agtggatgtt tgggggcaaa agaatgctgt caaaggtggc caatccaaca 1080

aatctcttag caattgttgg aaattttgta ggagccttat tgggtgcatc aatgggccta 1140

aaagaagggc ctcttttctt ctttgctctt gggcttgctc actacatggt gttgtttgta 1200

actctctccc agatgcttcc aacaaataag accatcccaa aagacctcca tccagtgttc 1260

tttctttttg tggcaccgcc tagtgttgct gctatggcat gggctaagat tcagggttca 1320

tttcattatg aatcaaggat tttctatttc actgccatgt tcctatatat ttcactggct 1380

gtccgggtca atcttttcag aggattcaaa ttctcacttt catggtgggc ctacactttt 1440

ccaatgactg ctgcagcaat tgctaccata acctatacaa atcaagtcac aaatgtacta 1500

actcaagctt tgagtgtaat attgagtctc attgctacat tcacagtaac agcagtgctt 1560

gtctcgacta tagtgcatgc ctttgtccta cgagacctct ttcccaatga ccttgccatt 1620

gccacaagtg agagaaagca aaaaccacgc aggaaatggc tccccattaa gacttgggag 1680

ccatga 1686

<210>2

<211>561

<212>PRT

<213> Artificial sequence

<220>

<223>

<400>2

Met Glu Asn Asn Leu Asn Arg Glu Thr Glu Gly His Gly Leu Pro Lys

1 5 10 15

Ile Pro Ser Leu Val Gln His Ile Ser Ser Asn Asp Glu Gly Asn Phe

20 25 30

Asp Asn Gly Asp Leu Lys Asn Leu Ser Ser Ser Phe Lys Glu Asn Glu

35 40 45

Thr Ile Leu Ala Gly Ser Gln Gly Asp Glu His Ala Ala Ile Asn His

50 55 60

Arg Asn Lys His Ser Val Ser Ile Asn Ile Pro Phe Ser Cys Glu Glu

65 70 75 80

Val Gln Leu His Asn Thr Val Arg Val Leu Tyr Ser Gly Glu Asn Asp

85 90 95

Phe Ser Ser Gln Ser Thr Thr Thr Thr Asp Ser Lys Pro Pro Leu Pro Ser

100 105 110

Gln Ser Met Pro Asn Gly Ser Leu His Ser Glu Pro Ala Ser Gly Val

115 120 125

Asn Phe Asn Asn Gln Glu Ser Val Ile Ser Leu Asn Asn Lys Arg Ile

130 135 140

Asp Phe Phe Lys Thr Trp Ser Ser Lys Leu Gly Gly His Ile Ser Val

145 150 155 160

Met Ser Gly Lys Val His Thr Glu Ser Ala Glu Asp Asp Asn Ser Leu

165 170 175

Cys Asn Thr Asn Lys Pro Leu Pro Val Asp Leu Phe Phe Lys Thr Leu

180 185 190

Glu Gly Pro Gln Leu Gln Thr Pro Lys Lys Ser Ser Glu Glu Met Val

195 200 205

Leu Pro Gln Asp Lys Gln Trp Pro Phe Leu Leu Arg Phe Pro Val Ser

210 215 220

Ser Phe Gly Ile Cys Leu Gly Val Ser Ser Gln Ala Ile Leu Trp Lys

225 230 235 240

Ala Leu Ala Thr Ser Pro Ser Thr Ala Phe Leu His Ile Thr Pro Lys

245 250 255

Ile Asn Phe Ile Leu Trp Phe Ile Ser Ile Gly Ile Val Ala Thr Ile

260 265 270

Phe Thr Thr Tyr Leu Phe Lys Ile Ile Leu His Phe Glu Ala Val Arg

275 280 285

Arg Glu Tyr Gln His Pro Val Arg Val Asn Phe Phe Phe Ala Pro Trp

290 295 300

Ile Ala Leu Leu Phe Leu Ala Leu Gly Val Pro Pro Ser Val Thr Lys

305 310 315 320

Asp Leu His Gln Ala Val Trp Tyr Ile Leu Met Ile Pro Leu Phe Cys

325 330 335

Leu Lys Leu Lys Ile Tyr Gly Gln Trp Met Phe Gly Gly Lys Arg Met

340 345 350

Leu Ser Lys Val Ala Asn Pro Thr Asn Leu Leu Ala Ile Val Gly Asn

355 360 365

Phe Val Gly Ala Leu Leu Gly Ala Ser Met Gly Leu Lys Glu Gly Pro

370 375 380

Leu Phe Phe Phe Ala Leu Gly Leu Ala His Tyr Met Val Leu Phe Val

385 390 395 400

Thr Leu Ser Gln Met Leu Pro Thr Asn Lys Thr Ile Pro Lys Asp Leu

405 410 415

His Pro Val Phe Phe Leu Phe Val Ala Pro Pro Ser Val Ala Ala Met

420 425 430

Ala Trp Ala Lys Ile Gln Gly Ser Phe His Tyr Glu Ser Arg Ile Phe

435 440 445

Tyr Phe Thr Ala Met Phe Leu Tyr Ile Ser Leu Ala Val Arg Val Asn

450 455 460

Leu Phe Arg Gly Phe Lys Phe Ser Leu Ser Trp Trp Ala Tyr Thr Phe

465 470 475 480

Pro Met Thr Ala Ala Ala Ile Ala Thr Ile Thr Tyr Thr Asn Gln Val

485 490 495

Thr Asn Val Leu Thr Gln Ala Leu Ser Val Ile Leu Ser Leu Ile Ala

500 505 510

Thr Phe Thr Val Thr Ala Val Leu Val Ser Thr Ile Val His Ala Phe

515 520 525

Val Leu Arg Asp Leu Phe Pro Asn Asp Leu Ala Ile Ala Thr Ser Glu

530 535 540

Arg Lys Gln Lys Pro Arg Arg Lys Trp Leu Pro Phe Lys Thr Trp Glu

545 550 555 560

Pro

Claims (9)

1. The application in regulating plant stress tolerance as described in any of the following; the stress tolerance is alkali resistance; The alkali resistance is resistance to carbonate stress; 1) protein, the amino acid sequence is the protein shown in sequence 2; 2) A nucleic acid molecule encoding the protein described in 1); 3) An expression cassette containing the nucleic acid molecule described in 2); 4) A recombinant vector containing the nucleic acid molecule described in 2); 5) A recombinant vector containing the expression cassette described in 3); 6) Recombinant microorganisms containing the nucleic acid molecules described in 2); 7) A recombinant microorganism containing the expression cassette described in 3); 8) A recombinant microorganism containing the recombinant vector described in 4); 9) A recombinant microorganism containing the recombinant vector described in 5). 2. The application of any one of the following in cultivating transgenic plants with increased stress tolerance; the stress tolerance is alkali resistance; The alkali resistance is resistance to carbonate stress; 1) protein, the amino acid sequence is the protein shown in sequence 2; 2) A nucleic acid molecule encoding the protein described in 1); 3) An expression cassette containing the nucleic acid molecule described in 2); 4) A recombinant vector containing the nucleic acid molecule described in 2); 5) A recombinant vector containing the expression cassette described in 3); 6) Recombinant microorganisms containing the nucleic acid molecules described in 2); 7) A recombinant microorganism containing the expression cassette described in 3); 8) A recombinant microorganism containing the recombinant vector described in 4); 9) A recombinant microorganism containing the recombinant vector described in 5). 3. Application in plant breeding as described below; 1) protein, the amino acid sequence is the protein shown in sequence 2; 2) A nucleic acid molecule encoding the protein described in 1); 3) An expression cassette containing the nucleic acid molecule described in 2); 4) A recombinant vector containing the nucleic acid molecule described in 2); 5) A recombinant vector containing the expression cassette described in 3); 6) Recombinant microorganisms containing the nucleic acid molecules described in 2); 7) A recombinant microorganism containing the expression cassette described in 3); 8) A recombinant microorganism containing the recombinant vector described in 4); 9) A recombinant microorganism containing the recombinant vector described in 5). 4. The application according to any one of claims 1-3, characterized in that: 2) the coding sequence of the nucleic acid molecule is a cDNA molecule or a genomic DNA molecule shown in sequence 1. 5. A method for cultivating a transgenic plant that stress tolerance improves, comprising improving the expression of the protein described in claim 1 in a recipient plant to obtain the step of a transgenic plant; the stress tolerance of the transgenic plant is higher than that of the transgenic plant The recipient plant; the stress tolerance is alkali resistance; The alkali resistance is resistance to carbonate stress. 6. The method according to claim 5, characterized in that: the stress tolerance of the transgenic plant is higher than that of the recipient plant in any of the following (1)-(5): (1) The seed germination rate of transgenic plants is higher than that of recipient plants; (2) The root length of the transgenic plant is longer than that of the recipient plant; (3) The above-ground biomass of the transgenic plant is higher than that of the recipient plant; (4) The chlorophyll content of the transgenic plant is higher than that of the recipient plant; (5) The electrolyte extravasation rate of transgenic plants was lower than that of recipient plants. 7. The method according to claim 5 or 6, characterized in that: The method for increasing the expression level of the protein described in claim 1 in the recipient plant is to overexpress the protein described in claim 1 in the recipient plant. 8. The method according to claim 5 or 6, characterized in that: The method for said overexpression is to introduce the coding gene of the protein described in claim 1 into the recipient plant; The nucleotide sequence of the gene encoding the protein is the DNA molecule shown in Sequence 1. 9. The method according to claim 5 or 6, characterized in that: the recipient plant is a monocotyledonous plant or a dicotyledonous plant.