CN117024547A

CN117024547A - GsSYP51b protein and application of encoding gene thereof in regulation and control of stress tolerance of plants

Info

Publication number: CN117024547A
Application number: CN202311130264.4A
Authority: CN
Inventors: 孙明哲; 孙晓丽; 贾博为; 崔红丽; 才晓溪; 王研; 关永旭
Original assignee: Heilongjiang Bayi Agricultural University
Current assignee: Heilongjiang Bayi Agricultural University
Priority date: 2023-09-04
Filing date: 2023-09-04
Publication date: 2023-11-10

Abstract

The application discloses an application of GsSYP51b protein and a coding gene thereof in regulating and controlling plant stress tolerance. The GsSYP51b protein is a 1) or a 2) or a 3) or a 4): a1 Amino acid sequence is a protein shown in sequence 3; a2 A fusion protein obtained by ligating a tag to the N-terminus or/and the C-terminus of the protein represented by the sequence 3; a3 A protein related to plant stress tolerance obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 3; a4 90% identity to the amino acid sequence shown in sequence 3, a protein derived from soybean and associated with plant stress tolerance. Experiments prove that the GsSYP51b gene is overexpressed in the arabidopsis thaliana, so that the tolerance of the arabidopsis thaliana to saline-alkali stress can be enhanced, and the protein can lay a foundation for the research of cultivating saline-alkali tolerant transgenic plants.

Description

GsSYP51b protein and application of encoding gene thereof in regulation and control of stress tolerance of plants

Technical Field

The application belongs to the technical field of biology, and particularly relates to an application of GsSYP51b protein and a coding gene thereof in regulating and controlling plant stress tolerance.

Background

Soil salinization seriously affects crop yield, and is one of the challenges of huge economic loss faced by realizing sustainable development worldwide. Meanwhile, saline-alkali adversity severely restricts the agricultural development in northeast areas of China and even nationwide, and is a great problem faced by the agricultural development in the adversity ecological area of China. It is counted that there are 8.31 hundred million hm worldwide ² Wherein, the saline-alkali soil of China accounts for about 10 percent. Along with the continuous growth of population and the degradation of the tillable soil in China, the method reasonably develops and utilizes the saline-alkali soil, excavates the agricultural production potential of the saline-alkali soil, and has important significance for increasing the tilling area in China, solving the grain problem and developing sustainable agriculture.

Along with the high-speed development of functional genomics and molecular biology, the technology of molecular design breeding is used for enhancing the salt and alkali tolerance of crops and improving the crop yield of saline-alkali soil, and the technology has become one of means for improving and reasonably developing and utilizing the saline-alkali soil. However, the important premise of realization is that the key salt and alkali tolerant regulatory genes with obvious functions are mined, and the salt and alkali stress signal transduction pathway and the salt and alkali tolerant molecular mechanism are clarified.

Disclosure of Invention

The application aims to solve the technical problem of how to regulate and control the stress tolerance of plants.

In order to solve the technical problems, the application firstly provides a novel application of the plant stress tolerance related protein GsSYP51b.

The application provides the application of GsSYP51b protein in the following 1) -3):

1) Regulating and controlling plant stress tolerance;

2) Cultivating transgenic plants with improved stress tolerance;

3) Plant breeding;

the GsSYP51b protein is a 1) or a 2) or a 3) or a 4):

a1 Amino acid sequence is a protein shown in sequence 3;

a2 A fusion protein obtained by ligating a tag to the N-terminus or/and the C-terminus of the protein represented by the sequence 3;

a3 A protein related to plant stress tolerance obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 3;

a4 90% identity to the amino acid sequence shown in sequence 3, a protein derived from soybean and associated with plant stress tolerance.

Wherein, sequence 3 consists of 251 amino acid residues.

The protein of a 2), wherein the tag refers to a polypeptide or protein which is fused and expressed together with the target protein by using a DNA in vitro recombination technology, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.

The protein according to a 3) above, wherein 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 not more than 10 amino acid residues or a substitution and/or deletion and/or addition of not more than 9 amino acid residues or a substitution and/or deletion and/or addition of not more than 8 amino acid residues or a substitution and/or deletion and/or addition of not more than 7 amino acid residues or a substitution and/or deletion and/or addition of not more than 6 amino acid residues or a substitution and/or deletion and/or addition of not more than 5 amino acid residues or a substitution and/or deletion and/or addition of not more than 4 amino acid residues or a substitution and/or deletion and/or addition of not more than 3 amino acid residues or a substitution and/or deletion and/or addition of not more than 2 amino acid residues or a substitution and/or deletion and/or addition of not more than 1 amino acid residue.

The protein according to a 4) above, wherein the identity is the identity of an amino acid sequence. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity. Such identity includes amino acid sequences having 90% or more, or 91% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more homology to the amino acid sequences shown in sequence 3 of the present application.

The protein described in the above a 1), a 2), a 3) or a 4) can be synthesized artificially or can be obtained by synthesizing the coding gene and then biologically expressing.

In order to solve the technical problems, the application also provides a new application of the biological material related to the GsSYP51b protein.

The application provides the application of the biological material related to GsSYP51b protein in the following 1) -3):

1) Regulating and controlling plant stress tolerance;

2) Cultivating transgenic plants with improved stress tolerance;

3) And (5) plant breeding.

The biomaterial is any one of the following A1) to A8):

a1 A nucleic acid molecule encoding a GsSYP51b protein;

a2 An expression cassette comprising A1) said nucleic acid molecule;

a3 A) a recombinant vector comprising the nucleic acid molecule of A1);

a4 A recombinant vector comprising the expression cassette of A2);

a5 A) a recombinant microorganism comprising the nucleic acid molecule of A1);

a6 A) a recombinant microorganism comprising the expression cassette of A2);

a7 A) a recombinant microorganism comprising the recombinant vector of A3);

a8 A recombinant microorganism comprising the recombinant vector of A4).

In the above applications, the nucleic acid molecule of A1) is a gene represented by the following B1) or B2) or B3) or B4):

b1 A genomic DNA molecule represented by SEQ ID NO. 1;

b2 A cDNA molecule represented by SEQ ID No. 2;

b3 A cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in B1) or B2) and encoding a GsSYP51B protein;

b4 Under stringent conditions with a nucleotide sequence defined by B1) or B2) or B3), and a cDNA molecule or genomic DNA molecule encoding the GsSYP51B protein.

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

The nucleotide sequence encoding the GsSYP51b protein of the application may be easily mutated by a person skilled in the art using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence encoding the GsSYP51b protein are derived from the nucleotide sequence of the present application and are equivalent to the sequence of the present application as long as they encode the GsSYP51b protein and have the same function.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of a protein consisting of the amino acid sequence shown in the coding sequence 3 of the present application. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.

The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

In the above application, the stringent conditions are hybridization and washing the membrane 2 times at 68℃in a solution of 2 XSSC, 0.1% SDS for 5min each time, and hybridization and washing the membrane 2 times at 68℃in a solution of 0.5 XSSC, 0.1% SDS for 15min each time; alternatively, hybridization and washing of the membrane were performed at 65℃in a solution of 0.1 XSSPE (or 0.1 XSSC) and 0.1% SDS.

In the above applications, the expression cassette (GsSYP 51b gene expression cassette) comprising a nucleic acid molecule encoding a protein GsSYP51b as described in A2) refers to DNA capable of expressing the protein GsSYP51b in a host cell, which DNA may include not only a promoter for initiating transcription of the GsSYP51b but also a terminator for terminating transcription of the GsSYP51b. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present application include, but are not limited to: a constitutive promoter; 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.

Recombinant vectors containing the GsSYP51b gene expression cassette can be constructed using existing expression vectors. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb, etc. The plant expression vector may also comprise the 3' -terminal untranslated region of a foreign gene, i.e. comprising a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylation to the 3 '-end of the mRNA precursor, and the untranslated regions transcribed from the 3' -end of, for example, the Agrobacterium tumefaciens induction (Ti) plasmid genes (e.g., nopaline synthase gene Nos) and plant genes (e.g., soybean storage protein genes) all have similar functions. When the gene of the present application is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene. To facilitate identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to the herbicide phosphinothricin, hph gene conferring resistance to antibiotic hygromycin, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or chemical marker genes, etc. (such as herbicide resistance genes), mannose-6-phosphate isomerase gene providing mannose metabolization ability, etc. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene.

In the above applications, the vector may be a plasmid, cosmid, phage or viral vector.

In the above application, the microorganism may be a yeast, a bacterium, an alga or a fungus, such as Agrobacterium.

In the above application, the stress tolerance may be resistance to salt and alkali stress.

Further, the salt and alkali stress tolerance may be carbonate stress tolerance.

Further, the carbonate stress tolerance may be NaHCO tolerance ₃ Stress.

In the application, the regulation of plant stress tolerance is to improve plant stress tolerance; the improvement of plant stress tolerance is reflected in: in NaHCO ₃ Under stress treatment, the higher the GsSYP51b protein content and/or activity in the plant or the higher the GsSYP51b gene expression level, the higher the stress tolerance of the plant; the method is further characterized in that: in NaHCO ₃ Under stress treatment, the higher the GsSYP51b protein content and/or activity or the higher the GsSYP51b gene expression level in the plant, the higher the seed germination rate, the higher the leaf expanding rate, the higher the fresh weight (fresh weight of leaves) of the overground part, the longer the root length and the better the growth vigor.

In such applications, the purpose of the plant breeding is to develop saline-alkali tolerant plants (e.g., carbonate tolerant plants).

In the above application, the plant is a monocotyledonous plant or a dicotyledonous plant, which can 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).

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

The method for cultivating transgenic plants with improved stress tolerance comprises the steps of improving the content and/or activity of GsSYP51b protein in a receptor plant to obtain transgenic plants; the transgenic plant is stress tolerant higher than the recipient plant.

In the above method, the stress tolerance may be salt and alkali stress tolerance.

Still further, the carbonate stress tolerance may be NaHCO tolerance ₃ Stress.

Further, the NaHCO-resistant ₃ Stress can be NaHCO resistance in germination period ₃ Stress or seedling stage NaHCO resistance ₃ Stress.

In a specific embodiment of the application, the NaHCO-resistant ₃ Stress is embodied in any one of the following X1) -X4):

x1) stress on carbonate (e.g. NaHCO) ₃ Stress), the seed germination rate of the transgenic plant is higher than the recipient plant;

x2) on carbonate stress (e.g. NaHCO) ₃ Stress), the transgenic plant exhibits a higher leaf expansion rate than the recipient plant;

x3) on carbonate stress (e.g. NaHCO) ₃ Stress), the transgenic plant has a root length longer than the recipient plant;

x4) on carbonate stress (e.g. NaHCO) ₃ Stress), the aerial parts of the transgenic plants have a higher fresh weight (leaf fresh weight) than the recipient plants.

The NaHCO ₃ The stress may specifically be 7mM NaHCO ₃ Or 9mM NaHCO ₃ 。

In the above method, the method for increasing the content and/or activity of the GsSYP51b protein in the recipient plant is to overexpress the GsSYP51b protein in the recipient plant.

Further, the over-expression method is to introduce the gene encoding the GsSYP51b protein into a recipient plant.

Furthermore, the coding gene of the GsSYP51b protein is shown as a sequence 2 in a sequence table.

In the above method, the recipient plant is a monocotyledonous plant or a dicotyledonous plant, which can be specifically 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).

In the above method, the transgenic plant is understood to include not only the first generation transgenic plant obtained by transforming the GsSYP51b gene into a recipient plant, but also the progeny thereof. For transgenic plants, the gene may be propagated in that species, and may be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, calli, whole plants and cells.

According to the application, the GsSYP51b gene is overexpressed in Arabidopsis thaliana, so that the GsSYP51b transgenic Arabidopsis thaliana is obtained. Experiments prove that: under carbonate stress, the seed germination rate and leaf expansion rate, root length and leaf fresh weight of GsSYP51b transgenic arabidopsis thaliana are higher than those of the recipient plants. The GsSYP51b gene can enhance the tolerance of arabidopsis to saline-alkali stress, and the GsSYP51b protein can lay a foundation for the research of cultivating saline-alkali tolerant transgenic plants.

Drawings

FIG. 1 shows subcellular localization analysis of GsSYP51b protein in plant cells.

FIG. 2 is a PCR identification of GsSYP51b transgenic glufosinate resistant seedlings.

FIG. 3 shows the RT-PCR detection of GsSYP51b transgenic Arabidopsis thaliana.

FIG. 4 is a germination carbonate tolerance analysis of GsSYP51b transgenic Arabidopsis thaliana.

FIG. 5 is a seedling stage carbonate tolerance analysis of GsSYP51b transgenic Arabidopsis.

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The soybean materials G07256 in the examples described below are described in the literature "Ge Y, li Y, zhu YM, bai X, lv DK, guo DJ, ji W, cai H: global transcriptome profiling of wild soybean (Glycine soja) roots under NaHCO treatment [ J ]. BMC plant biology 2010,10 ] and" Ge Ying, zhu Yanming, lv Dekang, dong Tingting, wang Weishi, tan-Shang-in, liu Caihong, ping. Study of wild soybean alkali stress reaction [ J ]. Grass science, 2009,26 (02): 47-52 ], which is available from the university of agricultural reclamation, and which is used only for the relevant experiments of the present application and is not used for other purposes.

pCAMBIA330035Su in the examples described below is described in the literature "Xiaoli Sun, wei Ji, xiaodong Ding, xi Bai, huan Cai, shanshan Yang, ue Qian, mingzhe Sun, yanming Zhu. 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", available to the public from the university of Heilongjiang, which is used only for the relevant experiments for the repeated application, and not as other uses.

pCAMBIA330035Su-NYFPu in the examples described below is described in the literature "Bowei Jia, mingzhe Sun, huizi Duanmu, xiaodong Ding, beidong Liu, yanming Zhu, xiaoli Sun. GsCHX19.3, a member of cation/H ⁺ exchanger superfamily from wild soybean contributes to high salinity and carbonate alkaline tolerance. Sci Rep 2017,7 (1): 9423", available to the public from the university of Gekko eight-farming, the biological material was used only for repeated experiments related to the application and was not used for other purposes.

Agrobacterium tumefaciens LBA4404 in the examples described below is described in the literature "Chen C, sun X, duanmu H, zhu D, yu Y, cao L, liu A, jia B, xiao J, zhu Y.GsCML27, a Gene Encoding a Calcium-Binding Ef-Hand Protein from Glycine soja, plays Differential Roles in Plant Responses to Bicarbonate, salt and genetic stress, PLoS one.2015Nov9;10 (11) e0141888.doi: 10.1371/journ.fine.0141888.PMID: 26550992; PMCID PMC4638360 ", available to the public from the university of October Heilongjiang, the biological material was used only for repeated experiments related to the present application and was not used for other purposes.

Example 1 acquisition of carbonate-related Gene GsSYP51b in Soybean

1. Basic information of a wild soybean SYP51b gene is downloaded through a Phytozome online database, and a Primer Premier 5.0 is used for designing a gene specific Primer with Bgl II and Pac I restriction enzyme recognition sequences according to a vector and a gene sequence as follows:

GsSYP51b-S：5'-ATAGATCTGATGCTGGCGCACTATAA-3' (underlined as Bgl II recognition sequence);

GsSYP51b-AS：5'-GGTTAATTAACAAATATTTGACCAGCAGC-3' (underlined is the Pac I recognition sequence).

The primer sequences were synthesized and prepared as 100. Mu. Mol/L stock solution using a concentration of 10. Mu. Mol/L and stored at-20 ℃.

2. Full, spot-free 3-week-old wild soybean G07256 seedlings were selected and total RNA was extracted by Trizol.

3. The first strand cDNA synthesis was performed using 5X HiScript III RT SuperMix (Vazyme).

4. Taking soybean total cDNA as a template, adopting high-fidelity DNA polymerase KOD One ^TM PCR amplification is carried out on PCR Master Mix-Blue (TOYOBO) to obtain a GsSYP51b gene full-length CDS region, and the nucleotide sequence of the region is shown as a sequence 2 in a sequence table. The full-length CDS region of the GsSYP51b gene was ligated with pEASY-T vector, positive clones were identified, submitted to company sequencing, and the correctly sequenced vector was designated pEASY-GsSYP51b for subsequent study.

EXAMPLE 2 subcellular localization analysis of GsSYP51b protein in plant cells

1. Based on USER ^TM Cloning techniques, the upstream primer GsSYP51b-U-S containing the GGCTTAAU linker and the downstream primer GsSYP51b-U-AS containing the GGTTTAAU linker were designed. The primer sequences were as follows:

GsSYP51b-U-S：GGCTTAAUATGCTGGCGCACTATAAT；

GsSYP51b-U-AS：GGTTTAAUTTACAAATATTTGACCAGCAGC。

2. PCR amplification was performed using the pEASY-GsSYP51b vector of example 1 as a template and the primers of step 1 to obtain the full-length CDS region of the GsSYP51b gene.

3. And (3) connecting the full-length CDS region of the GsSYP51b gene obtained in the step (2) with a Pac I and Nt.BbvC I double-digested pCAMBIA230035Su-NYFPu empty vector by using a USER enzyme (NEB, M5505S), and constructing a pCAMBIA230035Su-NYFPu-GsSYP51b vector.

4. The pCAMBIA230035Su-NYFPu-GsSYP51b vector is transformed into agrobacterium, and the transient expression of tobacco leaves is further adopted to analyze the positioning of GsSYP51b protein in cells, and the expression position of yellow fluorescent protein is observed through a laser confocal microscope.

The results are shown in FIG. 1, which shows: since NyFPu-GsSYP51b has yellow light around cells, that is, on plasma membranes, it is assumed that GsSYP51b protein is localized to the plasma membranes.

Example 3 acquisition of GsSYP51b transgenic Arabidopsis plants and saline-alkali tolerance analysis

1. Acquisition of GsSYP51b transgenic Arabidopsis plants

1. Based on USER ^TM The pCAMBIA330035Su vector of the cloning technology is used as a plant over-expression vector. The pCAMBIA330035Su vector was double digested with restriction enzymes Pac I and Nt.BbvC I to obtain a linearized vector.

2. The pEASY-GsSYP51b vector of example 1 was used as a template in KOD One ^TM PCR Master Mix-Blue (TOYOBO) high-fidelity DNA polymerase was used to perform PCR amplification using the primers of example 2 to obtain the gene sequence of GsSYP51b (HA-GsSYP 51 b) with an HA tag at the N-terminus.

3. The obtained linearization vector, HA-GsSYP51b and USER enzyme (NEB, M5505S) are incubated for 20min at 37 ℃, uracil of the GsSYP51b gene fragment is cut by using the USER enzyme to form a sticky end which can be complementary with the pCAMBIA330035Su vector, then the mixture is incubated for 20min at 25 ℃, and E.coli competent cells DH5 alpha are transformed, plasmids are extracted from positive transformants are activated, and the plasmids are submitted to sequencing analysis by a company, so that no frame shifting, mismatch and the like are ensured in the process of amplifying the gene by PCR, and the recombinant expression vector pCAMBIA330035Su-GsSYP51b is obtained.

Sequencing results showed that: the recombinant expression vector pCAMBIA330035Su-GsSYP51b is a vector obtained by inserting a DNA molecule shown in a sequence 2 between two PacI cleavage sites of the pCAMBIA330035Su vector and keeping other sequences of the pCAMBIA330035Su vector unchanged.

4. The recombinant expression vector pCAMBIA330035Su-GsSYP51b is transformed into the agrobacterium tumefaciens LBA4404 by adopting a freeze thawing method, the colony PCR is identified as positive, the successful transfer of the recombinant vector into the agrobacterium tumefaciens is indicated, and the positive recombinant bacterium is marked as pCAMBIA330035Su-GsSYP51b/LBA4404.

5. The recombinant bacterium pCAMBIA330035Su-GsSYP51b/LBA4404 is infected with wild Arabidopsis thaliana (Columbia ecology type) by adopting an agrobacterium-mediated inflorescence infection method, so that the GsSYP51b gene is over-expressed in the Arabidopsis thaliana. T harvesting the above ₀ After the transgenic arabidopsis seeds are disinfected by NaClO, the transgenic arabidopsis seeds are inoculated on a 1/2MS solid screening medium containing 25mg/L glufosinate. Transferring the screened out excessive expression transgenic Arabidopsis thaliana with glufosinate resistance into soil for culture, marking the Arabidopsis thaliana as different strains, and harvestingWhen seeds are planted, single plants are collected.

6. The genomic DNA of wild type Arabidopsis thaliana (WT) and transgenic Arabidopsis thaliana with glufosinate resistance were extracted using the full-length gold EasyPure genomic DNA extraction kit, and PCR detection was performed using specific primers used for full-length cloning of the GsSYP51b gene. At the same time with ddH ₂ O was used as a negative control and the positive plasmid pCAMBIA330035Su-GsSYP51b was used as a positive control.

The results are shown in fig. 2, which shows that: with ddH ₂ O as a template and the negative control with wild type Arabidopsis as a template had no amplified band, while other samples amplified bands consistent with the positive plasmid size, indicating that the exogenous gene GsSYP51b had been integrated into the Arabidopsis genome.

7. Each T obtained in the step 6 ₀ And carrying out reproduction on the generation positive plants, and repeating 2-3 generations of screening to obtain the transgenic arabidopsis homozygous plant line with glufosinate resistance. Extracting total RNA of wild arabidopsis (WT) and transgenic arabidopsis homozygous strain plants, taking reverse transcription products diluted 5 times as templates, adopting specific primers to carry out RT-PCR detection, and taking an action 2 gene as an internal reference.

The results are shown in fig. 3, which shows that: under the condition that the brightness of amplified bands of the action 2 genes is consistent, the WT plant cDNA is used as a template, no amplified bands are used, and the GsSYP51b transgenic arabidopsis homozygous lines can amplify target bands, and the sizes of the bands are consistent. The exogenous gene GsSYP51b is not only successfully integrated into the Arabidopsis genome, but also can be normally transcribed and expressed in transgenic Arabidopsis. They can be used in subsequent experiments. Selecting T ₃ The transgenic arabidopsis thaliana homozygous lines #2, #4 and #6 of the generation GsSYP51b were used for the carbonate resistance analysis described below.

2. GsSYP51b transgenic Arabidopsis carbonate tolerance assay

1. Carbonate tolerance analysis of GsSYP51b transgenic Arabidopsis during germination

Selection of full and consistent vigor wild type Arabidopsis thaliana (WT) and T ₃ Seed of transgenic arabidopsis thaliana homozygous lines #2, #4 and #6 of the generation GsSYP51b were sterilized under sterile conditions with 5% NaClO for 5min and sterilizedddH ₂ Repeatedly washing O to remove NaClO, placing in a refrigerator at 4deg.C for vernalization for 2d, and sowing in normal 1/2MS culture medium and containing 7mM or 9mM NaHCO ₃ 1/2MS medium of (C) and culturing at 22 ℃ for 10-14d. Observing seedling growth vigor, counting seed germination rate within 0-6d, and counting leaf expansion rate after 14d. Experiments were repeated three times, with 30 plants per treatment line.

The results are shown in fig. 4, which shows: in a normal 1/2MS culture medium, each strain germinates and grows normally, and the germination rate and seedling growth vigor are not different. However, through NaHCO ₃ After treatment, seed germination was inhibited in both wild-type and GsSYP51b transgenic Arabidopsis lines, but seed germination was higher in GsSYP51b transgenic Arabidopsis lines than in wild-type Arabidopsis, especially via NaHCO ₃ After 14d stress treatment, the growth of all plants was inhibited compared to plants under normal growth conditions, but the leaf expansion rate of the GsSYP51b transgenic arabidopsis lines was significantly higher than that of wild type arabidopsis, indicating that the ability of the GsSYP51b transgenic arabidopsis lines to withstand carbonate stress during germination was higher than that of wild type arabidopsis.

2. Seedling stage carbonate tolerance analysis of GsSYP51b transgenic Arabidopsis

Selecting wild Arabidopsis thaliana (WT) and T after NaClO sterilization ₃ Seed of transgenic arabidopsis thaliana homozygote strain #2, #4 and #6 of generation GsSYP51b, sowing in normal 1/2MS solid culture medium, culturing for 6d, transplanting arabidopsis thaliana seedlings with consistent growth vigor in normal 1/2MS culture medium and containing 7mM NaHCO respectively ₃ Is vertically cultured on 1/2MS culture medium for 6-10d. After 10d cultivation, root length and fresh weight of aerial parts (fresh weight of leaves) were measured, and the experiment was repeated three times, and 30 plants were used for each treatment line.

The results are shown in fig. 5, which shows that: through NaHCO ₃ After treatment, all plants were inhibited from growing, while the seedling vigor of the GsSYP51b transgenic arabidopsis line was significantly better than that of wild type arabidopsis, and root length and aerial part fresh weight (leaf fresh weight) were significantly higher than that of wild type arabidopsis, indicating that the ability of the GsSYP51b transgenic arabidopsis line to withstand carbonate stress during seedling stage was higher than that of wild type arabidopsis.

In conclusion, the excessive expression of the GsSYP51b gene obviously improves the carbonate stress tolerance of arabidopsis, and the GsSYP51b protein can positively regulate and control the salt and alkali tolerance of plants.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims

Use of the gssyp51b protein in 1) -3) as follows:

1) Regulating and controlling plant stress tolerance;

2) Cultivating transgenic plants with improved stress tolerance;

3) Plant breeding;

the GsSYP51b protein is a 1) or a 2) or a 3) or a 4):

a1 Amino acid sequence is a protein shown in sequence 3;

a2 A fusion protein obtained by ligating a tag to the N-terminus or/and the C-terminus of the protein represented by the sequence 3;

a3 A protein related to plant stress tolerance obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 3;

a4 90% identity to the amino acid sequence shown in sequence 3, a protein derived from soybean and associated with plant stress tolerance.
2. Use of a biological material related to the GsSYP51b protein in the following 1) -3):

1) Regulating and controlling plant stress tolerance;

2) Cultivating transgenic plants with improved stress tolerance;

3) Plant breeding;

the biomaterial is any one of the following A1) to A8):

a1 A nucleic acid molecule encoding a GsSYP51b protein;

a2 An expression cassette comprising A1) said nucleic acid molecule;

a3 A) a recombinant vector comprising the nucleic acid molecule of A1);

a4 A recombinant vector comprising the expression cassette of A2);

a5 A) a recombinant microorganism comprising the nucleic acid molecule of A1);

a6 A) a recombinant microorganism comprising the expression cassette of A2);

a7 A) a recombinant microorganism comprising the recombinant vector of A3);

a8 A recombinant microorganism comprising the recombinant vector of A4).
3. The use according to claim 2, characterized in that: a1 The nucleic acid molecule is a gene represented by the following B1) or B2) or B3) or B4):

b1 A genomic DNA molecule represented by SEQ ID NO. 1;

b2 A cDNA molecule represented by SEQ ID No. 2;

b3 A cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in B1) or B2) and encoding the GsSYP51B protein as defined in claim 1;

b4 Under stringent conditions with a nucleotide sequence defined in B1) or B2) or B3), and a cDNA molecule or a genomic DNA molecule encoding a GsSYP51B protein as described in claim 1.
4. Use according to any one of claims 1-4, characterized in that: the stress tolerance is salt and alkali stress tolerance.
5. A method of growing a transgenic plant with increased stress tolerance comprising the steps of: increasing the content and/or activity of the GsSYP51b protein of claim 1 in a recipient plant to obtain a transgenic plant; the transgenic plant is stress tolerant higher than the recipient plant.
6. The method according to claim 5, wherein: the stress tolerance is salt and alkali stress tolerance;

and/or, the transgenic plant has a stress tolerance higher than that of the recipient plant, embodied in any one of the following X1) -X4):

x1) seed germination of the transgenic plant under carbonate stress is higher than that of the recipient plant;

x2) the leaf expansion rate of the transgenic plant is higher than that of the recipient plant under carbonate stress;

x3) the root length of the transgenic plant is longer than the recipient plant under carbonate stress;

x4) the fresh weight of the aerial parts of the transgenic plant is higher than the recipient plant under carbonate stress.
7. The method according to claim 5, wherein: the method for increasing the content and/or activity of the GsSYP51b protein according to claim 1 in a recipient plant is to overexpress the GsSYP51b protein in the recipient plant.
8. The method according to claim 7, wherein: the over-expression method is to introduce the encoding gene of the GsSYP51b protein into a receptor plant.
9. The method according to any one of claims 5-8, wherein: the coding gene sequence of the GsSYP51b protein is shown as a sequence 2 in a sequence table.
10. The use according to any one of claims 1-4 or the method according to any one of claims 5-9, characterized in that: the plant is a dicotyledon or monocotyledon;

and/or, the dicotyledonous plant is a plant of the family Brassicaceae;

and/or, the crucifer is arabidopsis thaliana.