WO2012083545A1

WO2012083545A1 - Oilseed rape vascular bundle specific promoter bnvsp and use thereof

Info

Publication number: WO2012083545A1
Application number: PCT/CN2010/080193
Authority: WO
Inventors: 王劲; 左开井; 徐东; 代其林
Original assignee: Wang Jin; Zuo Kaijing; Xu Dong; Dai Qilin
Priority date: 2010-12-23
Filing date: 2010-12-23
Publication date: 2012-06-28

Abstract

An oilseed rape vascular bundle specific promoter BnVSP is disclosed by the present invention. The BnVSP promoter is useful for specifically expressing drought resistance and salt tolerance genes in plant vascular bundles by recombinant technology.

Description

Rape vascular bundle-specific expression promoter BnVSP and its application

Technical field

The present invention is in the field of biology, and in particular, the present invention relates to a promoter isolated from a Brassica napus genome using molecular cloning techniques, and its expression characteristics are confirmed by transgenic means. The invention also relates to the use of the promoter for improving drought resistance and salt tolerance of plants.

Background technique

Salt stress and drought severely affect plant growth. In order to improve the drought resistance and salt tolerance of crops, it is necessary to improve the osmotic adjustment ability of plants and reduce the toxicity of metal ions.

The absorption of metal ions and water by plants is mainly carried out through vascular bundles and conduits. The role of the conduits is to absorb water, while the role of vascular bundles is to absorb metal ions (Na ⁺ , K ⁺ ) and nutrients (such as nitrogen, phosphorus, etc. required by plants). ). Therefore, the expression of specific proteins in the vascular bundle of plants can promote the water absorption of plants and reduce the absorption of Na ⁺ , thereby reducing the content of toxic ions in plant cells and improving the drought resistance and salt tolerance of plants.

To date, the CaMV35S promoter (tobacco mosaic virus promoter) has been used to increase the salt tolerance and percolation resistance of plants. Since CaMV35S is a constitutively expressed promoter, over-expressed target genes may result in a decrease in plant yield or quality. Therefore, it is critical to clone a suitable promoter to enable the expression of the gene of interest in a target tissue (such as a catheter or a sieve) to achieve gene function.

Therefore, there is an urgent need to develop new promoters, especially promoters capable of realizing the localization and expression of genes in plant vascular bundles, and to achieve the purpose of improving plant drought resistance and salt tolerance.

Summary of the invention

It is an object of the present invention to provide a novel promoter and fragment analog thereof which enables localized expression of a gene in a plant vascular bundle.

Another object of the present invention is to use the promoter for improving drought and salt tolerance of plants.

The present inventors firstly isolated the BnVSP promoter from the constructed rape genomic DNA by molecular cloning method, and confirmed by experiments that it has vascular bundle-specific expression characteristics, the DNA molecule and the target gene (such as aquaporin, nitrogen transporter gene). And the terminator is ligated to form an expression cassette. The transgenic plants obtained by the expression cassette can realize the localized expression of genes in the plant vascular bundle, and transport the water and nutrients (such as Κ ⁺ , Ρ0 ₄ ³ -, and NO ³ ) in the soil to various tissues and organs of the plant (such as Leaves, flowers, fruits) improve the drought and salt tolerance of plants. The present invention has been completed on this basis. In a first aspect of the invention, a novel gene was discovered, designated BnVSP, which has the nucleotide sequence set forth in SEQ ID NO: 1.

The present invention also provides an isolated BnVSP promoter comprising the above BnVSP gene or a conservative variant thereof.

In a second aspect of the invention, a method for improving drought resistance and salt tolerance of a plant is provided, comprising the steps of:

(a) ligating the BnVSP promoter with a drought-tolerant and salt-tolerant gene (such as an aquaporin gene) to construct a plant expression vector;

(b) the vector obtained above is transferred into the Agrobacterium system to obtain Agrobacterium carrying the plant expression vector of the BnVSP promoter;

(c) contacting the plant cell or tissue or organ with the Agrobacterium obtained in the step (b), thereby transferring the BnVSP gene into the plant cell and integrating it into the chromosome of the plant cell; the Agrobacterium may be selected from EHA105.

(d) selecting a plant cell or tissue or organ into which the BnVSP gene is transferred;

(e) Regenerating plant cells or tissues or organs obtained in step (d) to obtain transgenic drought-tolerant and salt-tolerant plants driven by the BnVSP promoter.

Other aspects of the invention will be apparent to those skilled in the art from this disclosure.

The nucleotide of the present invention is a DNA molecule which can control the specific expression of a gene in a plant vascular bundle. The DNA molecule is ligated to a known drought-tolerant salt-tolerant gene (such as aquaporin, sodium-inverting protein gene, etc.) and a terminator to form an expression cassette. The transgenic plants obtained by the expression cassette can realize the localized expression of the target gene in the plant vascular bundle, and transport the water and nutrients (such as K ⁺ , Ρ 0 ₄ ³ - , and NO ³ - ) in the soil to the tissues and organs of the plant ( Such as leaves, flowers, and fruits) improve the drought and salt tolerance of plants.

In the present invention, the terms "BnVSP" and "BnVSP promoter" are used interchangeably and both refer to a BnVSP nucleotide sequence (SEQ ID NO: 1).

As used herein, "isolated" means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment). For example, the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is separated and purified, such as from other substances existing in the natural state. .

The polynucleotide of the present invention may be in the form of DNA. DNA forms include genomic DNA or synthetic DNA.

DNA can be single-stranded or double-stranded. The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 70%, preferably at least 80% identity between the two sequences. The invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions. In the present invention, "stringent conditions" means: (1) hybridization and elution at a lower ionic strength and a higher temperature, such as 0.2 X SSC, 0.1% SDS, 60 °C; or ( 2) Hybridization is carried out with a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum/0. l% Fi col l, 42 °C, etc.; or (3) only in two sequences Hybridization occurs when the identity between them is at least 90% or more, more preferably 95% or more. Furthermore, the hybridizable polynucleotide coding sequence has the same biological function as that shown in SEQ ID NO: 1.

The BnVSP nucleotide sequence of the present invention or a fragment thereof can usually be obtained by artificial synthesis or PCR amplification. For example, full sequence synthesis is first performed according to the sequence of SEQ ID NO: 1.

For the PCR amplification method, primers can be designed according to the nucleotide sequence disclosed in the present invention, and the artificially synthesized BnVSP nucleotide full-length sequence or a fragment thereof can be used as a template to amplify the relevant sequence.

In the present invention, the BnVSP polynucleotide sequence can be inserted into a recombinant expression vector to control the expression of other genes. The term "recombinant expression vector" refers to bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In summary, any plasmid and vector can be used as long as it can replicate and stabilize in the host. An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.

Methods well known to those skilled in the art can be used to construct expression vectors containing BnVSP promoter driven functional genes and suitable transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The DNA sequence can be operably linked to a multiple cloning site in an expression vector to direct mRNA synthesis of the functional gene. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

The appropriate sequence comprising the above can be used to transform a suitable host cell to enable it to express a protein. The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells; insect cells, and the like.

When the polynucleotide of the present invention drives expression of a gene of interest in higher eukaryotic cells, the transcriptional activity of the promoter is enhanced if an enhancer sequence is inserted into the vector. An enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, acting on a promoter to enhance transcription of the gene.

It will be apparent to one of ordinary skill in the art how to select appropriate vectors, genes, enhancers and host cells. Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl ₂ method, and the procedures used are well known in the art. Another method is to use MgCl ₂ . Conversion can also be carried out by electroporation if desired. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like. Transformed plants can also be subjected to methods such as Agrobacterium transformation or gene gun transformation, such as the leaf disc method. For transformed plant cells, tissues or organs, plants can be regenerated by a conventional method to obtain plants having improved drought resistance and salt tolerance.

A part or all of the polynucleotide of the present invention can be immobilized as a probe on a microarray or a DNA chip (also referred to as "gene chip") for detecting the presence of the sequence.

The BnVSP promoter of the present invention has the following characteristics: A) The function is clear, the promoter can drive a reporter gene (such as a fluorescent reporter gene, etc.) to be specifically expressed in the vascular bundle of the plant; B) the structure is new, at the nucleic acid level and has been seen The reported vascular bundle-specific promoter does not have any homology, and the homology site does not contain existing patent protection sequences and mutation sites.

The BnVSP promoter provides a new way to achieve transgene localization expression, and thus has great application prospects. By linking the BnVSP promoter to the target gene and introducing it into crop varieties (such as rice, wheat, corn, rapeseed, and cotton), the drought and salt tolerance levels of existing excellent crop varieties can be changed, and drought-resistant and salt-tolerant crop varieties can be obtained. Practical problems in agricultural production.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press 1989), or according to the manufacturer. Recommended conditions. BRIEF DESCRIPTION OF THE DRAWINGS:

Figure 1: Plant expression vector for BnVSP promoter and reporter gene; (LB: left border of T-DNA; polyA: terminator; Kan: kanamycin resistance gene; CaMV35S: tobacco mosaic virus promoter; BnVSP: Promoter; GFP/GUS: reporter gene; NOS: N0S terminator; RB: right border of T-DNA.)

Figure 2: PCR detection of transgenic BnVSP promoter seedlings (T.) leaves. Μ is DL2000 Marker: 1000, 750, 500, 250 and 100 bp; BnVSP is a positive control (plasmid); WT is a non-transgenic rapeseed; 1-7 is a transgenic rapeseed seedling.

Figure 3: GUS staining effect of C ^J^<3⁄43⁄4 carrier rapeseed; (AB is the whole plant dyeing effect; C: flower; D: staining concentrated on vascular bundle; D: leaf vascular bundle staining; E: apical staining Situation.) Figure 4: BnVSP promoter drives a plant expression vector for aquaporin gene expression; (LB: left border of T-DNA; polyA: terminator; Kan: kanamycin resistance gene; CaMV35S: tobacco mosaic virus promoter; BnVSP: promoter; Aquaporin: aquaporin; GFP/GUS: reporter gene; NOS: N0S terminator; The right border of T-DNA. ) detailed description

Example 1 DNA fragment cloning of the BnVSP promoter

(1) Extraction of rapeseed DNA

The specific steps for rapid extraction of rapeseed genomic DNA are as follows:

1) Take 100 mg of rapeseed fresh leaves and rapidly grind to powder in liquid nitrogen, add 400 P L DNA extraction buffer and 6 L RNase A (10 mg/μL), vortex for 1 min, and place in 60 ° water bath for 20-30 min. .

2) Add 130 P of buffer trichloromethane and mix well.

3) Centrifuge at 12000 r/min for 5 min and transfer the supernatant to a new centrifuge tube.

4) Repeat step 3.

5) Add 2 volumes of isopropanol to the supernatant and mix well. At this point, flocculent genomic DNA will appear.

6) Carefully pick up the flocculent DNA pellet with a pipette tip and rinse 3-5 times with 700 w L70% ethanol.

7) Remove excess ethanol and water and allow the DNA to air dry at room temperature.

8) Add appropriate amount of elution buffer TE, dissolve the DNA in a water bath at 65 °C for 10 minutes, and then mix and dissolve several times to help dissolve the solution. Finally, obtain the DNA solution and store at -20 °C for later use.

(2) DNA fragment cloning of the BnVSP promoter

The rape genomic DNA was diluted to 0.1 _μg / μL. The genomic DNA was digested according to the following system: The following components were added to a 1.5 mL tube: 25 genomic DNA, 80 U endonuclease (Dral, EcoRV, Pvul, Stu I), 10 L 10 X buffer, 57 μL ddH ₂ 0.

The solution was gently mixed first, and then the tube was placed in a 37 ° C water bath and digested overnight. 95 P L of phenol was added to each tube and mixed for 10 seconds. Then, the tube was centrifuged at 12000 r/min for 5 min, and the supernatant was transferred to a new centrifuge tube.

190 μL of ice-cold 95% ethanol and 9.5 μL of 3Μ NaAc (pH 5. 2) were added to each tube. The solution in the test tube was mixed and centrifuged at 4 ° C, HOOO rpm for 15 min. The supernatant was discarded and the precipitate was washed by adding 100 μL of ice-cold 80% ethanol. After washing, the pellet was centrifuged at 14,000 rpm for 10 min, the supernatant was discarded and the precipitate was dried. DNA was dissolved by adding a 20 μ LTE solution (ρ Η 7.5).

The DNA was ligated to the adaptor according to the Taraka kit. Specific ligation reaction systems include: 4 μL DNA, 1.9 μL Genome Walker adaptor, 1.6 μL Ligase Buffer, 0.5 L DNA ligase. The mixture was ligated overnight at 16 degrees.

PCR amplification was performed according to the instructions of the Advantage 2 Polymerase Mix Kit (Clontech).

First round of PCR system: 40 μL ddH20, 5 u L10X Advantage 2 PCR Buffer, 1 μL dNTP (10 mM each) , 1 μL API (10 μ M), 1 μL Advantage 2 Polymerase Mix (50X), 1 μ L GSP1 (10 μΜ) , 1 PLDNA. PCR procedure: 7 cycles 94 °C, 25s, 72 °C, 3min _; 32 cycles 94°C, 25s; 67°C, 3min _; one cycle 67°C, 7min„

The product of the first round of PCR reaction was diluted 2 μL into 98 μL dd3⁄40. Take 1 μL as a substrate for the second round of PCR amplification.

Second round of PCR system: 40 μL ddH20, 5 μL 10X Advantage 2 PCR Buffer, 1 dNTP (10 mM each) , 1 μL AP2 (10 μ M), 1 μL Advantage 2 Polymerase Mix (50X), 1 μL GSP2 (10 μΜ), 1 μ LDNA. PCR procedure: 7 cycles 94 ° C, 25 s, 72 ° C, 3 min _; 32 cycles 94 ° C, 25 s; 67 ° C, 3 min; - a cycle of 67 ° C, 7 min.

The amplified PCR product was electrophoresed on a 1% agarose gel. The recovered PCR was used directly for the analysis of the sample. The BnVSP promoter sequence was obtained after the analysis was correct.

Example 2 Artificial Synthesis of BnVSP Promoter Fragments

Based on the completed nucleotide sequence containing the 557p coding region, a single-stranded oligonucleotide fragment having a cohesive end of about 150-200 bp in length was synthesized from the positive and the sub-chain sequences, respectively, in four segments. Four complementary single-stranded oligonucleotide fragments each corresponding to each other in the forward and the secondary strands were annealed to form four double-stranded oligonucleotide fragments with sticky ends. Mixed-duplex oligonucleotides, by T ₄ DNA ligase catalyzed BnVSP assembled into a complete promoter. The synthetic DNA fragment contains the nucleotide sequence of SEQ ID NO: 1, and both ends of the synthetic gene contain Xbal and NC0I sites.

The artificially synthesized 5' and 3' end cleavage sites were used as Xbal and Ncol sites BnVSP promoter for the construction of plant expression vectors for BnVSP functional analysis below.

Construction of plant expression vector for BnVSP promoter and reporter gene The specific method for constructing the plant expression vector of the BnVSP promoter and the reporter gene is as follows:

1) BnVSP promoter and pCAMBIA1304 f Xbal and Ncol were double-digested to recover the DNA fragment after digestion;

2) The above-described digested fragments were ligated to each other to obtain BnVSP-driven GFP and GUS plant expression vectors (Fig. 1). It was then transferred to Agrobacterium LBA4404 for transformation of canola.

Example 4 Agrobacterium-mediated genetic transformation to obtain transgenic rapeseed plants

The steps of obtaining transgenic plants obtained by using Agrobacterium-mediated genetic transformation to obtain transgenic rapeseed plants are as follows:

1) Obtaining sterile seedlings: The rapeseed seeds are first soaked in sterile water for 10 minutes, then disinfected with 75% alcohol for 1 min, then rinsed with sterile water for 2 to 3 times. Disinfect with 10% NaC10 for 12 min, rinse with sterile water for 2 to 3 times, and inoculate on hormone-free MS medium. Sterile canola seedlings were used for transformation after 5 days of light culture.

2) Pre-culture: The cotyledons and growth points were cut out, and hypocotyls of about 0.5 cm long at the growth point were taken and cultured on a preculture medium for 2 days.

3) Culture of Agrobacterium: Add LBA4404 bacterial solution to 50 ml of Agrobacterium oxysporum culture medium (containing kanamycin, rifampicin and streptomycin 50 mg/L), shake at 220 rpm, shake at 28 °C for about 16 hr; At room temperature 4000 rpm, lOmin, discard the supernatant, the cells were suspended in MS liquid medium (including AS lOOuM), diluted to 50 times the original volume, and cultured for 1 hr under the same conditions as above, so that the bacteria solution was 0D600= 0. 5 or so.

4) Co-cultivation: The hypocotyls pre-cultured for 2 days were placed in the bacterial solution for 1 min, and then taken out and placed on a preculture medium covered with a sterile filter paper for 2 days (dark culture).

5) Selection culture: The hypocotyls after 2 days of co-culture were placed on the screening medium and subcultured every two weeks until emergence.

6) Rooting culture: Cut the regenerated shoots and place them on the rooting medium to root.

7) Transfer the transgenic plants with better roots directly into a small pot containing wet soil, and incubate in the culture chamber at 28 °C -30 °C (16h light / 8h dark, circulating light culture) for 2 weeks, then Transfer these small pots to the greenhouse. The transgenic plants are watered daily, fertilized, etc. until the rapeseed flowering seeds mature.

8) PCR identification of transgenic plants: Total plant DNA is obtained by minimizing the total DNA of plants, to 1. 5 μ 1 total

DNA was used as a template for PCR amplification with primers (corresponding to l-25bp, 530-550bp in sequence 1). A total of 126 sterile rapeseed plants were detected, and 12 of them were positive plants with specific bands (T The results of electrophoresis of PCR products of some plants are shown in Figure 2. Example 5 Tissue expression analysis of transgenic rapeseed plants;

GUS staining was carried out according to the method of Jeferson et al. (Jefferson, R. A., T. A. Kavanagh, and M. W. Bevan, GUS fusions: 5-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J, 1987. 6 : 3901-3907.). Specific steps are as follows:

Take the transgenic rape seedlings in the Eppendof tube and add the appropriate amount of GUS staining solution. The immersion material shall prevail. The tubes were placed at 37 ° C overnight. After 12 hours, the plants were blue. At this time, the GUS staining solution was decanted, and then decolorized twice by adding 70% ethanol for 12 h each time, and then the staining results were observed (Fig. 3).

Example 6 Using a Promoter to Directly Express Genes to Improve Drought and Salt Tolerance Characteristics of Rape

The specific method for constructing the plant expression vector of the BnVSP promoter and the aquaporin gene is as follows:

1. Construction of the vector: The plant expression vector DNA obtained in Example 3 was digested with Ncol and Pmll, and the digested DNA was subjected to electrophoresis to recover a large DNA fragment; the aquaporin gene was digested with Ncol and Pml1, and The large DNA fragment in the previous step was ligated, and the ligation product was transformed into E. coli cell DH5a to obtain an expression vector for the aquaporin gene replacement GFP-GUS gene (see Fig. 4). The obtained plasmid DNA was transferred into Agrobacterium strain EHA105, and the Agrobacterium was used to transform rapeseed.

2. Genetic transformation: Genetic transformation was carried out as described in Example 4.

3. Experiments on drought resistance and salt tolerance of transgenic plants

(1) Investigation on drought resistance and salt tolerance of transgenic rapeseed plants

Experimental methods: The transgenic rapeseed seeds and non-GM roasted rapeseed seeds identified as positive plants were immersed in a 2% sodium hypochlorite solution for 10 minutes, immersed in running water for 3-5 times, then swelled for 12 hours, and placed in a 25 ° C light incubator for germination. 24h, the germinated seeds were washed 2-3 times with distilled water. Seeds with uniform germination were inserted into a porcelain dish containing sterilized quartz sand, poured with appropriate amount of water, placed in a tissue culture room (25 ± 1 ° C, light for 12 h), and water was replenished twice a day. After the seedlings grew to 3-4 leaves, the plants were treated with different salt concentrations of rapeseed using 1/2MS solution containing 200 mmol / L NaCl.

Transgenic rapeseed plants and non-transgenic rapeseed plants were cultured in a medium containing 0匪ol and 250mmol NaCl to study the survival rate and development progress of the plants; the observations were observed on 5d, 10d and 15d. The transgenic rapeseed can grow normally on 250 匪ol of NaCl medium, and the non-transgenic rapeseed can not grow on 250 mmol of NaCl medium. The results show that the sequence is resistant to salt stress.

(2) Investigation on drought resistance and salt tolerance of transgenic rapeseed seeds experimental method:

Transgenic canola seeds treated with salt (200mm _O l /L NaCl) and non-transgenic rapeseed seeds that were not salt treated were broadcast live on October 15, 2009. The cell area is about 12 n, which is designed by random block, repeated 3 times, and routinely managed.

Before the maturity, 15 strains of normal growth were randomly selected from each plot for agronomic traits. The agronomic traits examined included plant height (cm), effective branch height (cm), main inflorescence length (cm), number of branches (number), number of effective pods per plant (number), and total yield per plant. Agronomic traits. The salt-treated transgenic plants were compared to non-transgenic non-transgenic rapeseed.

test results:

See Table 1. In the table, strains NO. 6, 10, 12, 17 are transgenic plants treated with 200 mM NaCl; Wtl6, 19, 8 are non-transgenic plants not treated with NaCl.

It can be seen from the table that the transgenic plants showed normal growth status even though they were treated with NaCl, and the agronomic traits of the transgenic plants were basically the same as those of the untreated non-transgenic plants.

Table 1 Comparison of agronomic traits between transgenic rapeseed plants and non-treated transgenic rapeseed after salt treatment

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it is to be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

Rights request

A BnVSP gene having the nucleotide sequence shown in SEQ ID NO: 1.

An isolated BnVSP promoter comprising the BnVSP gene of claim 1.

3. A vector comprising the BnVSP promoter of claim 2.

4. A genetically engineered host cell comprising the vector of claim 3.

5. A method for identifying a BnVSP promoter having a microtubule-specific expression characteristic, comprising:

(a) cultivating the host cell of claim 4 under conditions suitable for expression;

(b) The activity of the BnVSP promoter was identified by the method of GUS staining.

6. A method for improving drought and salt tolerance characteristics of a plant, comprising the steps of:

(a) ligating the BnVSP promoter of claim 2 with a drought-tolerant and salt-tolerant gene of interest to construct a plant expression vector;

(b) the obtained vector is transferred into an Agrobacterium system to obtain an Agrobacterium carrying a plant expression vector of the BnVSP promoter;

(c) contacting the plant cell or tissue or organ with the Agrobacterium obtained in step (b), thereby transferring the gene of claim 1 into the plant cell and integrating it into the chromosome of the plant cell;

(d) selecting a plant cell or tissue or organ that is transferred to the gene of claim 1;

(e) Regenerating the plant cell or tissue or organ obtained in the step (d) to obtain a transgenic drought-tolerant and salt-tolerant plant driven by the BnVSP promoter.

7. The method according to claim 6, wherein the Agrobacterium is EHA105.

8. The use of the BnVSP gene according to claim 1 for cultivating drought-tolerant and salt-tolerant plants.

9. Use of a promoter according to claim 2 for cultivating drought tolerant and salt tolerant plants.