Brassica napus Bna.A08. SNAT2 protein and application of coding gene thereof in salt stress resistance

Abstract

The invention discloses a brassica napus Bna.A08. SNAT2 protein and application of a coding gene of the brassica napus Bna.A08. SNAT2 protein in salt stress resistance, the Bna.A08. SNAT2 gene is introduced into brassica napus through a genetic engineering technology, and it is found that the tolerance of the brassica napus in a salt stress environment can be remarkably improved through expression of the gene. Bna.A08. SNAT2 protein is used as a key salt-tolerant factor, cells are protected from being damaged by salt stress by adjusting osmotic pressure and ion balance in oilseed rape cells, germination of oilseed rape under salt stress is promoted, cultivation and popularization of salt-tolerant crop varieties are expected to be promoted through further research and application, and the application prospect is wide. Powerful support is provided for solving the problems of saline-alkali soil utilization and grain safety.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of protein Bna.A08.SNAT2 and encoding genes in salt stress tolerance. Background Art

[0002] Rapeseed is the largest source of domestic edible vegetable oil, accounting for more than 55% of the domestic oil crop oil production, and is the core of maintaining the security of national edible oil supply. Soil salinization is one of the main abiotic stresses that endanger agricultural production. At present, about 20% of the world's arable land is harmed by salinization, and about 6.6% of my country's arable land is eroded by salinization. Due to factors such as climate change, unreasonable irrigation and excessive application of chemical fertilizers, the degree of secondary salinization of soil in my country has continued to increase, and the area of ​​salinized soil has increased, seriously endangering my country's agricultural production and national food security. Rapeseed is one of the salt-tolerant crops. It has strong tolerance whether in coastal saline-alkali land (mainly containing NaCl), inland saline-alkali land (mainly containing Na2SO4, Na2CO3), or soda saline-alkali land (mainly containing Na2CO3, NaHCO3). The key to genetic improvement of crop salt tolerance is the cloning and utilization of excellent salt-tolerant genes. Therefore, exploring rapeseed salt-tolerant genes and analyzing their functions are of great significance for breeding salt-tolerant rapeseed varieties and increasing rapeseed yields. Summary of the invention

[0003] The purpose of the present invention is to provide a protein Bna.A08.SNAT2 which affects the salt tolerance of plant seeds during germination period and its encoding gene.

[0004] The present invention provides a protein derived from Brassica napus L., named Bna.A08.SNAT2, which is the following protein (a1) or (a2);

[0005] (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing;

[0006] (a2) a protein derived from (a1) in which one or more amino acid residues of the amino acid sequence of sequence 1 in the sequence list are substituted and / or deleted and / or added and which is related to the salt tolerance of plants;

[0007] (a3) a protein having 75% or more homology with the amino acid sequence defined in Sequence 1 in the sequence list, derived from Brassica napus and associated with salt tolerance during seed germination;

[0008] In order to facilitate the detection of the protein in (a1), the amino terminus or carboxyl terminus of the protein shown in sequence 1 in the sequence listing may be connected to a tag as shown in Table 1.

[0009] Table 1. Sequences of tags

[0010] Label Residue sequence HA 9 YPYDVPDYA

[0011] The protein in (a2) above can be artificially synthesized, or its coding gene can be synthesized first and then expressed biologically; the coding gene of the protein in (a2) above can be obtained by deleting one or several codons of amino acid residues in the DNA sequence shown in positions 1-591 of sequence 2 in the sequence list, and / or performing missense mutations of one or several base pairs, and / or connecting the coding sequence of the tag shown in Table 1 to its 5' end and / or 3' end.

[0012] The present invention also protects the nucleic acid molecule encoding the protein Bna.A08.SNAT2.

[0013] The nucleic acid molecule encoding protein Bna.A08.SNAT2 may be a DNA as shown in (b1) or (b2) or (b3) or (b4) below;

[0014] (b1) the nucleotide sequence of which is the DNA molecule shown in Sequence 2 in the sequence listing;

[0015] (b2) a DNA molecule whose nucleotide sequence is the 1st to 591st position of Sequence 2 in the sequence list;

[0016] (b3) a DNA molecule that hybridizes with the DNA defined in (b1) or (b2) under stringent conditions, which is derived from Brassica napus and encodes the protein Bna.A08.SNAT2;

[0017] (b4) A DNA molecule derived from Brassica napus and having 75% or more homology with the DNA molecule defined in (b1) or (b2) and encoding the above protein.

[0018] 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;

[0019] Among them, sequence 2 in the sequence list consists of 591 deoxynucleotides and is the full-length cDNA sequence encoding the protein, of which 1-591 is the coding region.

[0020] The stringent conditions may be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M Na3PO4 and 1mM EDTA, and rinsing at 50°C in 2× nucleic acid hybridization treatment solution (SSC), 0.1% SDS. It may also be: hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and rinsing at 50°C in 1×SSC, 0.1% SDS. It may also be: hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and rinsing at 50°C in 0.5×SSC, 0.1% SDS. It may also be: hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and rinsing at 50°C in 0.5×SSC, 0.1% SDS. The membrane can be hybridized in a mixed solution of EDTA and rinsed in 0.1×SSC, 0.1% SDS at 50°C. It can also be hybridized in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA at 50°C and rinsed in 0.1×SSC, 0.1% SDS at 65°C. It can also be hybridized in a solution of 6×SSC and 0.5% SDS at 65°C and then washed once with 2×SSC, 0.1% SDS and once with 1×SSC, 0.1% SDS.

[0021] Those skilled in the art can easily mutate the nucleotide sequence encoding the protein Bna.A08.SNAT2 of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence of the protein Bna.A08.SNAT2 isolated from the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein Bna.A08.SNAT2.

[0022] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or higher identity to the nucleotide sequence of the protein Bna.A08.SNAT2 composed of the amino acid sequence shown in Sequence 1 in the coding sequence table of the present invention. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0023] The present invention also protects a recombinant vector, an expression cassette, a transgenic cell line, a recombinant bacterium or a recombinant virus containing any of the above-mentioned nucleic acid molecules.

[0024] Available existing plant expression vector construction contains the recombinant expression vector of described nucleic acid molecule.Described plant expression vector comprises binary agrobacterium vector and can be used for the vector of plant microprojectile bombardment etc.As pROKII, pBin438, pGreen, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pCAMBIA3301, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA company) etc.Described plant expression vector can also comprise the 3 ' end non-translated region of foreign gene, promptly comprise polyadenylic acid signal and any other DNA fragment that participates in mRNA processing or gene expression.Described polyadenylic acid signal can guide polyadenylic acid to join the 3 ' end of mRNA precursor, as agrobacterium crown gall induction (Ti) plasmid gene (as rouge synthase Nos gene), plant gene (as soybean storage protein gene) 3 ' end transcription non-translated region all has similar function. When the gene is used to construct a recombinant plant expression vector, any enhanced promoter (such as cauliflower mosaic virus (CAMV) 35S promoter, corn ubiquitin promoter (Ubiquitin)), constitutive promoter or tissue-specific expression promoter (such as seed-specific expression promoter) can be added before the transcription start nucleotide, which can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive and can be natural or synthetic. The translation start region can come from the transcription start region or the 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 that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, 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, the hph gene that confers resistance to the antibiotic hygromycin, and the dhfr gene that confers resistance to methatrexate, and the EPSPS gene that confers resistance to glyphosate) or chemical resistance marker genes (such as herbicide resistance genes), and mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose.

[0025] In the example of the present invention, the recombinant vector is pCAMBIA1300-35S-Bna.A08.SNAT2-6HA;

[0026] The recombinant vector pCAMBIA1300-35S-Bna.A08.SNAT2-6HA is obtained by inserting the nucleic acid molecule (the DNA fragment shown at positions 1-588 of sequence 2 in the sequence table) between the KpnI and SpeI restriction sites of pCAMBIA1300-35S-6HA.

[0027] The use of the above-mentioned protein or the above-mentioned nucleic acid molecule or the above-mentioned recombinant vector, expression cassette, transgenic cell line, recombinant bacteria or recombinant virus in regulating plant salt tolerance is also within the scope of protection of the present invention.

[0028] The use of the above protein or the above nucleic acid molecule or the above recombinant vector, expression cassette, transgenic cell line, recombinant bacteria or recombinant virus in cultivating salt-tolerant plants or plants with improved drought resistance is also within the scope of protection of the present invention.

[0029] Another object of the present invention is to provide a method for cultivating transgenic plants with improved salt tolerance.

[0030] The method provided by the present invention is as follows (1) or (2):

[0031] (1) The method steps are as follows: increasing the content and / or activity of any of the above-mentioned proteins in the target plant to obtain a transgenic plant with higher salt tolerance than the target plant;

[0032] (2) The method steps are as follows: increasing the expression level of the nucleic acid molecule encoding the above protein in the target plant to obtain a transgenic plant with higher salt tolerance than the target gene plant.

[0033] In the above method, the increasing of the content or activity of any of the above proteins Bna.A08.SNAT2 in the target plant, or the increasing of the expression of the nucleic acid molecule encoding the above protein in the target plant, can be achieved by methods well known in the art such as transgenic, multiple copies, changing promoters, regulatory factors, and introducing nucleic acid molecules encoding the protein into the target plant.

[0034] The present invention also protects a plant breeding method, comprising the following steps: increasing the content and / or activity of any of the above-mentioned proteins Bna.A08.SNAT2 in the plant, thereby increasing the salt tolerance of rapeseed seeds during the germination period.

[0035] Any of the plants described above may be any of the following c1) to c4): c1) dicotyledonous plants; c2) cruciferous plants; c3) Brassica napus; c4) Brassica napus variety 'K407'. The above-mentioned improvement in stress resistance may be manifested as an increase in germination rate, a decrease in at least one of hydrogen peroxide (H2O2) and malondialdehyde (MDA) content, and an increase in at least one of the antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT).

[0036] Experiments have shown that overexpression of the Bna.A08.SNAT2 gene in the Brassica napus variety 'K407' can improve the salt tolerance of Brassica napus, and the improved salt tolerance is manifested in an increased germination rate under salt stress, a reduced H2O2 and active oxygen content, and an increased antioxidant enzyme activity. The present invention proves that the protein Bna.A08.SNAT2 is of great significance in breeding and research to improve plant salt tolerance, and can be used to cultivate salt-tolerant plant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Analysis of the expression pattern of Bna.A08.SNAT2 gene under salt stress;

[0038] Figure 2 The subcellular localization of Bna.A08.SNAT2-GFP fusion protein;

[0039] Figure 3 The construction map of pCAMBIA1300-35S-Bna.A08.SNAT2-6HA expression vector, DNA identification gel image of overexpression material and the expression level of Bna.A08.SNAT2 gene in T3 generation Bna.A08.SNAT2 transgenic rapeseed;

[0040] Figure 4 This is the germination phenotype analysis of T3 generation Bna.A08.SNAT2 transgenic rapeseed under salt stress;

[0041] Figure 5 To analyze the contents of hydrogen peroxide (H2O2) and malondialdehyde (MDA) in T3 generation Bna.A08.SNAT2 transgenic rapeseed under salt stress;

[0042] Figure 6 The results are statistical analysis of the contents of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in T3 generation Bna.A08.SNAT2 transgenic rapeseed under salt stress. DETAILED DESCRIPTION

[0043] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0044] The complete steps of the method of the present invention are described

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0046] The materials, kits, enzymes, culture media, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

[0047] Example 1. Obtaining protein Bna.A08.SNAT2 and its encoding gene

[0048] 1. Cloning of protein Bna.A08.SNAT2 and its encoding gene

[0049] About 30 seeds of rapeseed variety 'K407' were taken and sown in plug trays with nutrient soil as the matrix (nutrient soil: vermiculite = 3:1), and cultured in an artificial climate culture room for 2 weeks; leaves were taken and quickly frozen in liquid nitrogen and ground, and total RNA of rapeseed leaves was extracted using an RNA extraction kit. After the extraction, the integrity of RNA was detected by agarose gel electrophoresis, and the RNA concentration and purity were also detected; then, the single-stranded cDNA of each RNA sample was synthesized by reverse transcription according to a cDNA synthesis kit, and then the concentration of the single-stranded cDNA was detected, and the single-stranded cDNA was synthesized. The cDNA concentration was diluted to 50 ng / μL; the cDNA was used as a template and PCR amplification was performed with BnaA08.CDS-F: 5'-ATGCTTCTCAGAGGCCCAATC-3' and BnaA08.CDS-R: 5'-TCACTTCTTGTTTCTCTGTTTACGC-3' as primers. The amplified product was subjected to agarose gel electrophoresis, and a 591 bp DNA fragment was separated and purified for sequencing. The results showed that the sequence of the DNA fragment was as shown in positions 1-591 of sequence 2 in the sequence list.

[0050] Sequence 2 in the sequence list is the full-length cDNA sequence encoding the protein Bna.A08.SNAT2 shown in sequence 1 in the rapeseed variety 'K407', wherein positions 1-591 are the coding sequence, and the cDNA does not contain the 5' non-coding region and the 3' non-coding region. The gene encoding the protein Bna.A08.SNAT2 is named gene Bna.A08.SNAT2.

[0051] 2. Gene expression analysis under salt stress during seed germination

[0052] 'K407' rapeseed seeds of uniform size were selected, disinfected with 75% alcohol twice, 1 min each time, and washed with distilled water 3 times, 2 min each time; then 7.5 mL of distilled water (control) or 7.5 mL of 200 mM NaCl solution was added to a culture dish covered with 3 layers of filter paper, and the culture dish was placed in a dark treatment at 4°C for 3 days, and then placed in a rapeseed growth room with a relative humidity of 54% and a temperature of 25°C (16 h light / 8 h dark) for normal culture, and the control and treated seeds of 0h, 2h, 6h, 12h, and 24h were taken and quickly frozen in liquid nitrogen; the above samples were ground according to the above method, total RNA was extracted, and reverse transcription was performed to obtain cDNA, and the above cDNA was used as a template to generate Bna.A08.qPCR-F: 5'-TCTCAGAGGCCCAATCTCCA-3' and Bn a.A08.qPCR-R: 5'-GAGTAGATGGGCGGTTGTCT-3' (amplify Bna.A08.SNAT2 gene) was used as the target gene primer, and BnGADPH-F: 5'-CCGCTTCCTTCAACATCATT-3' and BnGADPH-R: 5'-GTCGCAGCTTTCTCGAGTCT-3' were used as reference gene primers to perform real-time fluorescence quantitative PCR (RT-qPCR) to analyze the expression pattern of Bna.A08.SNAT2 gene under salt stress during seed germination.

[0053] The results are as follows Figure 1 As shown, the expression of Bna.A08.SNAT2 gene was upregulated by salt stress during seed germination.

[0054] Example 2: Subcellular localization of Bna.A08.SNAT2-GFP fusion protein

[0055] Using the cDNA of 'K407' as a template, PCR amplification was performed using primers Bna.A08.SNAT2-GFP-KpnI-F: 5'-GTCGACGGTATCGATAAGCTTATGCTTCTCAGAGGCCCAATC-3' and Bna.A08.SNAT2-GFP-SpeI-R: 5'-TCCCCCGGGCTGCAGGAATTCCTTCTTGTTTCTCTGTTTACG-3', and the target gene was connected into the expression vector pGreen-35S-GFP by homologous recombination. The nucleotide sequence in the overexpression vector pGreen-35S-Bna.A08.SNAT2-GFP is the sequence of positions 1-588 of the DNA of sequence 2 in the sequence table. The tobacco leaves were transformed by transient expression and observed under a laser confocal microscope.

[0056] like Figure 2As shown, the red signal is the nuclear marker signal mCherry and the endoplasmic reticulum marker signal ER, the blue signal is the chloroplast autofluorescence, and the pGreen-35S-Bna.A08.SNAT2-GFP fusion protein vector is distributed in the nucleus, cytoplasm and chloroplast, proving that the Bna.A08.SNAT2 protein is localized in the nucleus, cytoplasm and chloroplast.

[0057] Example 3: Overexpression of Bna.A08.SNAT2 gene to improve salt tolerance in rapeseed

[0058] 1. Construction of overexpression recombinant vector

[0059] Using the cDNA of 'K407' as a template, PCR amplification was performed with Bna.A08.SNAT2-6HA-F: 5'-TCTAGAGGATCCCCGGGTACCATGCTTCTCAGAGGCCCAATC-3' and Bna.A08.SNAT2-6HA-R: 5'-GTATGGGTAACTAGAACTAGTCTTCTTGTTTCTCTGTTTACG-3' as primers, and the target gene was connected into the expression vector pCAMBIA1300-35S-6HA between the KpnI and SpeI restriction sites (located downstream of the 35S promoter) by homologous recombination. After sequencing confirmation, the recombinant vector pCAMBIA1300-35S-Bna.A08.SNAT2-6HA was obtained, which expressed the Bna.A08.SNAT2 protein shown in Sequence 1.

[0060] 2. Genetic transformation, screening and identification of Brassica napus

[0061] The recombinant vector pCAMBIA1300-35S-Bna.A08.SNAT2-6HA was transformed into the Agrobacterium tumefaciens GV3101+pSoup19 strain to obtain the recombinant Agrobacterium containing the recombinant vector pCAMBIA1300-35S-Bna.A08.SNAT2-6HA. 2 to 3 days before transformation, the Agrobacterium containing the pCAMBIA1300-35S-Bna.A08.SNAT2-6HA overexpression vector obtained above was activated, and then the activated corresponding strain was inoculated into the Agrobacterium suspension and placed in a shaker for 30 minutes; the cultured bacterial solution was placed in a centrifuge tube, and the OD value of the Agrobacterium was measured with a spectrophotometer, and the OD 600The value is 0.1-0.2; the rape explants pre-cultured for 2-3 days are placed in the Agrobacterium suspension for infection for 10 minutes; the infected rape explants are placed on the filter paper to dry and placed on the co-cultivation medium; two days later, the explants are transferred to the extended screening medium for 7 days; the effective callus tissue is selected and transferred to the screening medium containing hygromycin for screening for about 15 days, and the screening is repeated 2-3 times; the vigorously growing positive callus tissue is transferred to the differentiation medium, and the seedlings are differentiated from the seedlings, and the differentiated seedlings are transferred to the rooting culture medium for rooting for 7-10 days. The positive seedlings formed by hygromycin selection, differentiation and rooting are defined as T1 transgenic plants.

[0062] The positive control pCAMBIA1300-35S-Bna.A08.SNAT2-6HA vector plasmid (Cas), wild-type material 'K407' and T1 generation overexpression transgenic plants were identified by PCR using p1300-F: 5'-GTCACTTTATTGTGAAGATAGTGG-3' and Bna.A08.SNAT2-6HA-R: 5'-GTATGGGTAACTAGAACTAGTCTTCTTGTTTCTCTGTTTACG-3' as identification primers.

[0063] 3. RT-qPCR detection of transgenic rapeseed

[0064] Detect the transcription level of the target gene in the T1 generation overexpressed transgenic plants. Total RNA was extracted from the leaves of the T1 generation overexpressed transgenic plants, and reverse transcription was performed to obtain cDNA. The above cDNA was used as a template, Bna.A08.SNAT2.qPCR-F: 5'-TCTCAGAGGCCCAATCTCCA-3' and Bna.A08.SNAT2.qPCR-R: 5'-GAGTAGATGGGCGGTTGTCT-3' (amplification of Bna.A08.SNAT2 gene) were used as target gene primers, and BnGADPH-F: 5'-CCGCTTCCTT CAACATCATT-3' and BnGADPH-R: 5'-GTCGCAGCTTTCTCGAGTCT-3' were used as reference gene primers for real-time fluorescence quantitative PCR (RT-qPCR) to detect the transcription level of the target gene in the T1 generation overexpressing transgenic plants. Then, four T1 generation positive seedlings with higher transcription levels were selected for further sowing and planting until all the offspring seedlings of a certain T2 generation offspring plant were identified to contain the target gene fragment. At this time, the seeds harvested from the offspring of the single plant were the T3 generation homozygous seeds, which were convenient for subsequent salt tolerance evaluation.

[0065] like Figure 3As shown, the expression levels of the target gene Bna.A08.SNAT2 in Bna.A08.SNAT2 transgenic rapeseed lines OE#1, OE#2, OE#3 and OE#4 were significantly higher than those in the wild-type 'K407' plant.

[0066] 4. Phenotypic analysis of salt stress tolerance in transgenic rapeseed seeds during germination

[0067] The two lines OE#1 and OE#2 with the highest expression of the target gene Bna.A08.SNAT2 among the Bna.A08.SNAT2 transgenic rapeseed lines OE#1, OE#2, OE#3 and OE#4 were selected as the subsequent experimental materials. Transgenic rapeseed plants of uniform size and rapeseed seeds of 'K407' were selected respectively, and three replicates were set for each treatment under normal conditions (distilled water) and stress treatment (200mM NaCl), and 50 seeds were used for each replicate. After the seeds were disinfected and washed with distilled water, they were placed in a 90mm diameter glass culture dish, 7.5mL of NaCl solution was added, and the culture dish was placed in a dark treatment at 4℃ for 3d, and then placed in a rapeseed growth room with a relative humidity of 54% and a temperature of 25℃ (16h light / 8h dark) for normal culture. The seed radicle breaking through the seed coat was used as the germination sign. The seed germination was observed at the same time point every day and the germination rate was counted. The germination was recorded by taking photos on the 8th day after stratification.

[0068] like Figure 4 As shown, the seed germination rates of Bna.A08.SNAT2 transgenic rapeseed lines OE#1 and OE#2 under salt stress during the germination period were significantly higher than that of the wild type 'K407'.

[0069] 5. Determination of physiological indicators of salt stress tolerance of transgenic rapeseed seeds during germination

[0070] The Bna.A08.SNAT2 transgenic rapeseed line and the wild-type 'K407' line were treated using the stress treatment method in the above 4, phenotypic analysis of salt stress tolerance during the germination period of transgenic rapeseed seeds, and samples were taken 6 hours after salt stress. The hydrogen peroxide content was determined using a hydrogen peroxide (H2O2) detection kit; the malondialdehyde content of the above materials was determined using a malondialdehyde (MDA) detection kit; the superoxide dismutase content was determined using a superoxide dismutase (SOD) detection kit; the peroxidase content was determined using a peroxidase (POD) detection kit; and the catalase content was determined using a catalase (CAT) content detection kit.

[0071] like Figure 5 As shown in the figure, the hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents of Bna.A08.SNAT2 transgenic rapeseed lines OE#1 and OE#2 were significantly lower than those of wild type 'K407' under salt stress during the germination period. Figure 6 As shown, under salt stress, the contents of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in Bna.A08.SNAT2 transgenic rapeseed lines OE#1 and OE#2 at the germination stage were significantly higher than those in the wild type 'K407'.

[0072] The results of Example 3 show that the protein Bna.A08.SNAT2 and its encoding gene have the function of regulating the salt tolerance of rapeseed. Overexpressing the encoding gene of protein Bna.A08.SNAT2 in plants can improve the salt tolerance of rapeseed.

Claims

1. A Brassica napus Bna.A08.SNAT2 protein, characterized in that: The protein has the function of improving the tolerance of Brassica napus under salt stress environment, and its amino acid sequence is shown in SEQ ID NO:

1.

2. The gene encoding the Brassica napus Bna.A08.SNAT2 protein according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

3. A method for improving the salt stress tolerance of Brassica napus, characterized in that: The method comprises introducing the gene of claim 2 into Brassica napus, allowing the gene to be expressed in Brassica napus, thereby improving the tolerance of Brassica napus under salt stress environment.

4. A recombinant vector containing the gene according to claim 2, characterized in that: The recombinant vector can express the gene in Brassica napus, thereby improving the tolerance of Brassica napus under salt stress environment.

5. A transgenic Brassica napus cell line containing the recombinant vector according to claim 4, characterized in that: The cell line can grow under salt stress environment and maintain a high survival rate and / or growth rate.

6. A method for cultivating salt stress tolerant Brassica napus, characterized in that: The method comprises introducing the recombinant vector of claim 4 into Brassica napus, obtaining transgenic Brassica napus plants by tissue culture technology, and selecting transgenic Brassica napus plants with improved salt stress tolerance for cultivation.

7. Use of the Brassica napus Bna.A08.SNAT2 protein according to claim 1 in improving the salt stress tolerance of plants.

8. Use of the gene encoding the Brassica napus Bna.A08.SNAT2 protein according to claim 2 in improving the salt stress tolerance of plants.

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