Application of BnaC04.bZIP16 gene in improving salt tolerance of plants

Abstract

The invention relates to the technical field of agricultural biology, in particular to application of a BnaC04.bZIP16 gene to improvement of salt tolerance of plants. The sequence of the BnaC04.bZIP16 gene provided by the invention is as shown in SEQ ID NO.1, and the coded protein sequence of the BnaC04.bZIP16 gene is as shown in SEQ ID NO.2. The invention discovers that the BnaC04.bZIP16 gene can be highly expressed after wild brassica napus is irrigated with saline water, and meanwhile, the transgenic brassica napus with the over-expressed gene can better grow in a salt stress environment, so that the BnaC04.bZIP16 gene can be used for preparing a salt-tolerant brassica napus. The rape with the BnaC04.bZIP16 gene knocked out is more difficult to grow in a salt stress environment, so that the effect of the BnaC04.bZIP16 gene as a forward regulatory factor in enhancing the salt tolerance of the rape is verified, and a key molecular tool is provided for developing a new variety of brassica napus with high salt tolerance; the gene has wide application potential and commercial value in the field of agricultural planting, especially in improving the salt tolerance of crops and cultivating transgenic crops with high salt tolerance.

Description

Technical Field

[0001] The present invention relates to the field of plant genetic engineering technology, in particular to Brassica napus BnaC04.bZIP16 Application of genes in enhancing plant tolerance to salt stress. Background Art

[0002] With global climate change and unreasonable land management practices, soil salinization has become one of the main abiotic adversities restricting agricultural production and crop growth. In addition to reasonable irrigation and drainage, soil improvement and other means, actively developing salt-tolerant crops through genetic engineering and other means is also an effective way.

[0003] As an important oil crop in China, the growth of Brassica napus is significantly affected by soil salinization. Soil salinization not only limits the growth of Brassica napus, but also affects its yield and quality. The middle and lower reaches of the Yangtze River are important production areas of Brassica napus in my country. The soil salt content is gradually increasing due to the lateral infiltration of seawater and the return of groundwater salt. The low-salt soil area is gradually expanding, affecting the yield of Brassica napus industry and other related crops. Therefore, it is urgent to cultivate Brassica napus suitable for growing in saline soil environments and to cultivate Brassica napus that can tolerate salt stress. Summary of the invention

[0004] The object of the present invention is to provide a BnaC04.bZIP16 Application of genes in improving salt tolerance in plants.

[0005] The present invention cloned the Brassica napus gene BnaC04.bZIP16 (BnaC04g09600D) and was found to play an important role in improving plant salt tolerance.

[0006] To achieve the present invention, in the first aspect, the present invention provides a Brassica napus gene BnaC04.bZIP16 Application in improving plant salt tolerance.

[0007] In the present invention, the amino acid sequence of the BnaC04.bZIP16 protein is at least one of the following: (1) As shown in SEQ ID NO.2.

[0008] (2) An amino acid sequence that has at least 90% homology to the amino acid sequence shown in SEQ ID NO. 2 and has an equivalent functional protein.

[0009] (3) A protein derived from SEQ ID NO. 2 by replacing, deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 2 and retaining the function of the amino acid sequence shown in SEQ ID NO. 2.

[0010] (4) An amino acid sequence obtained by connecting a tag, a restriction site and / or a connecting peptide sequence to the N-terminus and / or C-terminus of any of the amino acid sequences of (1) to (3).

[0011] In a second aspect, the present invention provides a Brassica napus BnaC04.bZIP16 The application of the gene in improving plant salt tolerance is characterized in that the BnaC04.bZIP16 The nucleotide sequence of the gene is at least one of the following: (1) As shown in SEQ ID NO.1.

[0012] (2) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO.1 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization at 65°C in a 0.1× SSPE solution containing 0.1% SDS or a 0.1× SSC solution containing 0.1% SDS, and the membrane is washed with the solution.

[0013] (3) A nucleotide sequence in which one or more nucleotides are substituted, deleted and / or added to the nucleotide sequence shown in SEQ ID NO.1 and the nucleotide sequence expresses a protein with the same functional properties.

[0014] (4) A nucleotide sequence that has more than 90% homology with the nucleotide sequence shown in SEQ ID NO.1 and expresses a protein with the same function.

[0015] The present invention found that plants will highly express BnaC04.bZIP16 The protein edited by the gene, and the plants that highly express the above protein transformed by genetic engineering means are also more adaptable to salt stress environment, while plants with the gene knocked out are more susceptible to salt stress environment, resulting in reduced yield.

[0016] In a third aspect, the present invention provides a method for improving plant salt tolerance, specifically, increasing the Brassica napus gene BnaC04.bZIP16 The expression level of the encoded protein in the plant, and the method includes but is not limited to the following.

[0017] (1) Overexpression through genetic engineering BnaC04.bZIP16 Gene vectors are transferred into plants; or

[0018] (2) Select and modify the above mentioned BnaC04.bZIP16 The promoter of the gene is a strong promoter; or

[0019] (3) Introduction of enhancers through genetic engineering; or

[0020] (4) Increasing the number of chromosomes in plants through genetic engineering BnaC04.bZIP16 Gene copy number.

[0021] In some embodiments, the BnaC04.bZIP16 The plasmid of the gene is transformed into plants and bred to homozygosity to obtain plants that overexpress the gene. BnaC04.bZIP16 In the present invention, Agrobacterium containing BnaC04.bZIP16 The plasmid of the gene was transformed into Brassica napus to obtain overexpression BnaC04.bZIP16 of genetically modified rapeseed.

[0022] In some embodiments, the breeding method can be any breeding method that can ultimately obtain homozygotes, including individual selection, haploid breeding, pedigree method, etc., preferably multi-generation breeding.

[0023] In a fourth aspect, the present invention provides a method for increasing the expression level of BnaC04.bZIP16 protein in plants, specifically, growing the corresponding plants in a salt stress environment, wherein the amino acid sequence of the BnaC04.bZIP16 protein is shown in SEQ ID NO.2, and growing in a salt stress environment can increase the expression level of the BnaC04.bZIP16 protein in the plants.

[0024] In a fifth aspect, the present invention provides Brassica napus BnaC04.bZIP16 Application of the gene, or the protein encoded by the gene, or the biological material containing the gene, or the method for improving plant salt tolerance in plant breeding; Brassica napus BnaC04.bZIP16 The nucleotide sequence of the gene is shown in SEQ ID NO.1; or

[0025] The amino acid sequence of the protein is shown in SEQ ID NO.2; The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector, engineered bacteria or non-renewable plant cells or tissues.

[0026] Preferably, the application in plant breeding is application in cultivating highly salt-tolerant plants or cultivating plants growing in a high-salt soil environment.

[0027] In a sixth aspect, the present invention provides Brassica napus BnaC04.bZIP16 Application of a gene, or a protein encoded by the gene, or a biological material containing the gene, or the above method for improving plant salt tolerance in increasing the fresh weight of plants under salt stress; Brassica napus BnaC04.bZIP16 The nucleotide sequence of the gene is shown in SEQ ID NO.1; or

[0028] The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0029] The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector, engineered bacteria or non-renewable plant cells or tissues.

[0030] In a seventh aspect, the present invention provides Brassica napus BnaC04.bZIP16 Application of the gene, or the protein encoded by the gene, or the biological material containing the gene, or the above-mentioned method for improving plant salt tolerance in genetic improvement of plant germplasm resources; Brassica napus BnaC04.bZIP16 The nucleotide sequence of the gene is shown in SEQ ID NO.1; or

[0031] The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0032] The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector, engineered bacteria or non-renewable plant cells or tissues.

[0033] Beneficial effects of the present invention: The present invention first discovered BnaC04.bZIP16 Gene has the function of improving plant salt tolerance and is overexpressed in plants BnaC04.bZIP16 The gene can significantly improve the growth ability of plants under salt stress, and plants with the gene knocked out are more difficult to grow in salt stress environments, indicating that BnaC04.bZIP16 Genes are of great significance in breeding for improving plant salt tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 In Example 2 of the present invention, the In-Fusion system is used. BnaC04.bZIP16 The CDS sequence of the recombinant plasmid was constructed on the entry vector pGWC pGWC-BnaC04.bZIP16 Schematic diagram of the structure.

[0036] Figure 2 In Example 2 of the present invention, the Gateway system is used to transfer the BnaC04.bZIP16 Recombinant plasmid constructed on plant expression vector pHZM137 pHZM137-BnaC04.bZIP16 Schematic diagram of the structure.

[0037] Figure 3 In Example 2 of the present invention, BsaI. Restriction endonucleases and T4 ligase are used to construct the gene editing vector containing the target sequence into the pHSE401 vector. pHSE401-giBnbZIP16 Structural diagram.

[0038] Figure 4 Wild type Westar rapeseed under 0.8% NaCl salt stress at different treatment times BnaC04.bZIP16 Schematic representation of the expression levels of . Error bars represent SD.

[0039] Figure 5 Wild-type rapeseed grown under 0.8% NaCl salt stress for 21 days, overexpressing BnaC04.bZIP16 Transgenic rapeseed and knockout genes BnaC04.bZIP16 Schematic diagram of the growth status and total fresh weight of rapeseed with genes. Note: WT in Figure A is wild-type rapeseed, OE is overexpressed BnaC04.bZIP16 Transgenic rapeseed, KO means knockout BnaC04.bZIP16 Rapeseed with genes; CK in Figure B is the wild-type rapeseed control, and OE is the overexpression BnaC04.bZIP16 Transgenic rapeseed, KO means knockout BnaC04.bZIP16 Genetic rapeseed. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Example 1 BnaC04.bZIP16 Gene cloning

[0042] Leaf RNA was extracted using the RNA extraction kit (Tiangen, DP432) from Tiangen Biochemical Co., Ltd. and reverse transcribed using the All-in-one RT Mix with gDNA Remover StarScript III one-tube genomic reverse transcription master mix (GenStar, A230). Follow the steps below.

[0043] RNA extraction: 1. Quickly freeze fresh Brassica napus leaves in liquid nitrogen and grind them. Add 500 μL SL (495 μL SL, 5 μL β-mercaptoethanol), shake and mix well. Centrifuge at 1,2000 rpm for 2 min at 4°C. Transfer 400 μL of supernatant to the CS column.

[0044] 2. Centrifuge at 4°C, 1,2000 rpm for 2 min, take the supernatant and transfer it to a new RNase-Free centrifuge tube. Add 180 μL of anhydrous ethanol, mix well, transfer to CR3, and centrifuge at 4°C, 12,000 rpm for 30 sec.

[0045] 3. Add 80 μL of DNase I working solution (10 μL DNase I storage solution, 70 μL RDD), and let stand at room temperature for 10 min.

[0046] 4. Add 350 μL of deproteinized solution RW1 and 500 μL of rinse solution RW, centrifuge at 12,000 rpm for 1 min. Place the CR3 column in a new RNase-Free centrifuge tube, add 30 μL of RNase-Free ddH2O, let stand at room temperature for 2 min, and centrifuge to obtain RNA solution.

[0047] Reverse transcription: 1. Prepare the reverse transcription system: Total RNA 0.1 μg - 1 μg, StarScript III All-in-one RTMix 1 μL, 5 × StarScript III All-in-one RT Buffer 4 μL, and RNase-free water to 20 μL.

[0048] 2. After mixing, use a PCR instrument to perform reverse transcription to obtain cDNA. PCR program: 37℃ 2 min, 50℃ 15 min, 85℃ 2 min.

[0049] Gene cloning: According to the rapeseed genome database sequence (https: / / yanglab.hzau.edu.cn / BnTIR), search BnaC04.bZIP16 CDS sequence, and then designed primer sequences with adapters and no stop codon (Forward: SEQ ID NO.3, Reverse: SEQ ID NO.4) to clone BnaC04.bZIP16 The CDS sequence was amplified by PCR using KOD high-fidelity enzyme and sequenced for identification. The gene sequence is shown in SEQ ID NO. 1 and the protein sequence is shown in SEQ ID NO. 2.

[0050] Example 2 BnaC04.bZIP16 Construction of overexpression and knockout vectors and transformation of rapeseed

[0051] Overexpression vector: Using the In-Fusion system, PCR products were connected to AhdI digested the entry vector pGWC, named pGWC-BnaC04.bZIP16 ( Figure 1 ), transformed into Escherichia coli DH5α, screened positive clones by PCR identification, and then constructed into the plant expression vector pHZM137 through the Gateway system and named pHZM137 - BnaC04.bZIP16 ( Figure 2 ), the correctly sequenced plasmid was transferred into GV3101 Agrobacterium to transform Brassica napus Westar (Genome-Widecharacterization of DGATs and their expression diversity analysis in response to abiotic stresses in Brassica napus . Yin, X., et al. 22 April 2022 Plants(Basel) 11(9): 1156.), and then multiply the seeds until they are pure and then harvest them for future use.

[0052] Knockout vector construction: Search on the CRISPR-GE website (http: / / skl.scau.edu.cn / home / ) BnaC04.bZIP16 Gene and its homologous genes BnaA05g08520D, BnaA03g56410D, BnaA04g20520D, design common gene editing target sites, design primers (Forward: SEQ ID NO.5, Reverse: SEQ ID NO.6), and construct them into the already Bsa Ⅰ digested pHSE401 The vector ( A CRISPR / Cas9 toolkit for multiplex genomeediting in plants. Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, WangXC, Chen QJ. BMC Plant Biol. 2014 Nov 29;14(1):327.10.1186 / s12870-014-0327-y) was named pHSE401-giBnbZIP16 ( Figure 3 ). Sequencing was performed using SEQ ID NO.7 and SEQ ID NO.8, and the plasmid with the correct sequencing was transferred into GV3101 Agrobacterium to transform Brassica napus, and the pure and harvested seeds were used for later use.

[0053] pHZM137-BnaC04.bZIP16 and pHSE401-giBnbZIP16The genetic transformation and screening of positive transgenic lines are as follows: (1) Disinfection: Soak full rapeseed Westar seeds in 75% alcohol for 1 min, disinfect them with 0.15% mercuric chloride solution for 12 min, wash them with sterile ddH2O for 5-6 times, sow them on M0 medium, and culture them in the dark at 22℃ in a culture room for 6-7 days.

[0054] (2) Infection: Plasmids pHZM137-BnaC04.bZIP16 or pHSE401-giBnbZIP16 After activation of Agrobacterium, shake the bacteria, collect the bacteria by centrifugation at 4000 r / min, suspend them in DM suspension and pour them into an empty culture dish for later use; use sterile tweezers and a scalpel to cut the hypocotyls of the seedlings, each with a length of 0.8 - 1.0 cm, place the cut explants in a dish with bacterial solution and immerse them for 10 min; dry the infected explants with sterile filter paper, transfer them to M1 culture medium, and culture them in the dark at 22℃ for 48 h.

[0055] (3) Callus induction: The hypocotyl explants after co-culture were transferred to M2 medium for callus induction and cultured under light for 20 days.

[0056] (4) Bud regeneration: Transfer the explants with normal growth and swelling at both ends to differentiation medium M3 and culture them under light. Subculture every 20 days until green buds appear.

[0057] (5) Rooting: The differentiated green shoots are transferred to M4 culture medium for growth, and subcultured every 20 days until roots grow. After roots grow, they are moved to flower pots to keep them moist and harden. After hardening is completed, they are transplanted to large pots for culture.

[0058] The screening antibiotic hygromycin B (Hyg, Roche, 200 μl / L) was added to M2, M3 and M4 culture media. After hardening, the overexpressing materials were sprayed with herbicide (Basta, Coolaber, 3 ml / L) for screening. The leaves of the knockout materials were taken to extract DNA, and sequencing was used to identify whether the knockout was successful.

[0059] Example 3 Wild-type rapeseed under salt stress BnaC04.bZIP16 Changes in gene expression levels

[0060] After the wild-type Westar cells were cultured under normal conditions for 5 days, they were irrigated with 0.8% NaCl solution for 0, 6, 12, and 24 hours. BnaC04.bZIP16 The expression level of the gene.

[0061] Fluorescence quantitative PCR was performed using 2 × RealStar Fast SYBR qPCR Mix (GenStar, A301) and the experimental procedures were performed according to the instructions.

[0062] 1. System: 2 × RealStar Fast SYBR qPCR Mix 10 μL, 1 μL of 10 μM upstream and downstream primers, 1 μL of cDNA, and fill to 20 μL with ddH2O.

[0063] 2. qPCR program: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 10 sec, annealing at 60°C for 30 sec, and amplification for 40 cycles.

[0064] 3. Data analysis used the double ΔCt method.

[0065] The results are as follows Figure 4 , at 6, 12 and 24 h under salt stress treatment, BnaC04.bZIP16 The expression of the gene was significantly increased compared with 0 hours, indicating that salt can induce BnaC04.bZIP16 Gene expression in plants.

[0066] Example 4 BnaC04.bZIP16 Analysis of the effect of genes on salt tolerance in rapeseed

[0067] 1. Filling soil: Fill a small red pot with a diameter of 17 cm with mixed soil (vermiculite: nutrient soil = 1:1) until the soil is level with the rim of the pot.

[0068] 2. Seed sowing: Select seeds with full grains and uniform size. BnaC04.bZIP16 Transgenic seeds (seeds of overexpression and knockout plants) and Westar seeds were sown in the soil. Three strains were selected for each transgenic material, and 10 seeds were sown for each strain, about 2 cm deep. After the cotyledons opened, there were 6 seedlings in each pot, and the excess were removed.

[0069] 3. Salt water irrigation: Prepare 0.8% NaCl solution, and water each small red pot with 800 mL for the first time to ensure that the water in each red pot does not leak out. Water once every five days in the later period, 100 mL each time. Adjust the time and amount of salt water irrigation according to the humidity, and treat with salt stress for 21 days.

[0070] 4. Biomass determination: After 21 days of salt stress, the total fresh weight (TFW) of rapeseed seedlings was measured.

[0071] like Figure 5 As shown, after 21 days of 0.8% NaCl stress, the overexpressing BnaC04.bZIP16 The fresh weight of the knockout plants (Table 1) was significantly higher than that of the wild type, increasing by about 23.5%, while the fresh weight of the knockout plants decreased significantly by 32.2%. This indicates that under salt stress conditions, BnaC04.bZIP16 It can improve the salt tolerance of Brassica napus.

[0072]

[0073] The present invention uses Brassica napus as an example to prove through experiments that BnaC04.bZIP16 The function of the gene helps the plant grow normally under salt stress.

[0074] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. An application of Brassica napus BnaC04.bZIP16 protein in improving plant salt tolerance, characterized in that: The amino acid sequence of the BnaC04.bZIP16 protein is at least one of the following: (1) as shown in SEQ ID NO.2; (2) an amino acid sequence that has at least 90% homology to the amino acid sequence shown in SEQ ID NO. 2 and has an equivalent functional protein; (3) a protein derived from SEQ ID NO. 2 and retaining the function of the amino acid sequence shown in SEQ ID NO. 2 by replacing, deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO. 2; (4) An amino acid sequence obtained by connecting a tag, a restriction site and / or a connecting peptide sequence to the N-terminus and / or C-terminus of any of the amino acid sequences of (1) to (3).

2. A Brassica napus BnaC04.bZIP16 The application of a gene in improving the salt tolerance of a plant is characterized in that: Said BnaC04.bZIP16 The nucleotide sequence of the gene is at least one of the following: (1) as shown in SEQ ID NO.1; (2) a nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO.1 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization in a 0.1× SSPE solution containing 0.1% SDS or a 0.1× SSC solution containing 0.1% SDS at 65°C and the membrane is washed with the solution; (3) a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or added to the nucleotide sequence shown in SEQ ID NO.1 and the nucleotide sequence expresses a protein with the same functional properties; (4) A nucleotide sequence that has more than 90% homology with the nucleotide sequence shown in SEQ ID NO.1 and expresses a protein with the same function.

3. A method for improving plant salt tolerance, characterized in that: Increase BnaC04.bZIP16 The expression level of a gene in a plant, and the method thereof includes but is not limited to: (1) Overexpression through genetic engineering BnaC04.bZIP16 The gene vector is transferred into the plant; or (2) Increase the amount of coding in plant chromosomes through genetic engineering BnaC04.bZIP16 The copy number of the gene; or (3) Introduction of enhancers through genetic engineering; or (4) Selection and modification through genetic engineering BnaC04.bZIP16 The promoter of gene expression is a strong promoter; Said BnaC04.bZIP16 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. The method according to claim 3, characterized in that will contain BnaC04.bZIP16 The gene plasmid is transformed into plants and the seeds are propagated until they are homozygous.

5. A method for increasing the expression of BnaC04.bZIP16 protein in plants, characterized in that: The corresponding plants are grown in a salt stress environment. The amino acid sequence of the BnaC04.bZIP16 protein is shown in SEQ ID NO.

2.

6. Brassica napus BnaC04.bZIP16 Use of a gene, or a protein encoded by the gene, or a biological material containing the gene, or a method according to any one of claims 3 to 5 in plant breeding; Brassica napus BnaC04.bZIP16 The nucleotide sequence of the gene is shown in SEQ ID NO.1; or The amino acid sequence of the protein is shown in SEQ ID NO.2; The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector, engineered bacteria or non-renewable plant cells or tissues.

7. The use according to claim 6, characterized in that: The application in plant breeding is to cultivate plants with high salt tolerance or to cultivate plants growing in a high-salt soil environment.

8. A Brassica napus BnaC04.bZIP16 Use of a gene, or a protein encoded by the gene, or a biological material containing the gene, or a method according to any one of claims 3 to 5 in increasing the fresh weight of plants under salt stress; Brassica napus BnaC04.bZIP16 The nucleotide sequence of the gene is shown in SEQ ID NO.1; or The amino acid sequence of the protein is shown in SEQ ID NO.2; The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector, engineered bacteria or non-renewable plant cells or tissues.

9. A Brassica napus L. BnaC04.bZIP16 Use of a gene, or a protein encoded by the gene, or a biological material containing the gene, or a method according to any one of claims 3 to 5 in genetic improvement of plant germplasm resources; Brassica napus BnaC04.bZIP16 The nucleotide sequence of the gene is shown in SEQ ID NO.1; or The amino acid sequence of the protein is shown in SEQ ID NO.2; The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector, engineered bacteria or non-renewable plant cells or tissues.

10. The use according to any one of claims 1-2, 6-9 or the method according to any one of claims 3-5, characterized in that: The plant is Brassica napus.

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