Application of brassica napus BnaC05.bZIP68 gene in improvement of plant salt tolerance

Abstract

The invention relates to the technical field of agricultural biology, in particular to application of a brassica napus BnaC05. BZIP68 gene to improvement of salt tolerance of plants. The sequence of the BnaC05.bZIP68 gene provided by the invention is as shown in SEQ ID NO.1, and the sequence of a protein coded by the BnaC05.bZIP68 gene is as shown in SEQ ID NO.2. The invention discovers that wild brassica napus can highly express the BnaC05.bZIP68 gene in a salt stress environment, and meanwhile, transgenic brassica napus overexpressed with the gene has stronger salt tolerance, so that the BnaC05.bZIP68 gene can be applied to the salt stress environment of the brassica napus. Therefore, the effect of the BnaC05. BZIP68 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 market value in the field of agricultural planting, especially in improving the salt tolerance of plants 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 BnaC05.bZIP68 Application of genes in improving salt tolerance in plants. Background Art

[0002] Soil salinization is a growing global problem that impedes plant growth and crop yields. An increasing number of studies have shown that genetic engineering plays an important role in helping plants cope with abiotic stresses such as salt stress.

[0003] Brassica napus Brassica napus L.) is one of the three major types of rapeseed. It is a composite species that evolved from amphidiploidization after natural interspecific hybridization between Chinese cabbage (AA, n = 10) and cabbage (CC, n = 9). It is native to Europe and is named for its leaf shape and plant type similar to cabbage. The chromosome group is AACC, n = 19. Cabbage rapeseed is the type with the highest grain yield among the three types of oil rapeseed (Brassica rapa, Brassica juncea, and Brassica oleracea). It is widely planted in the middle and lower reaches of the Yangtze River in my country and is an important oil crop in my country. However, the soil in some areas of the middle and lower reaches of the Yangtze River has gradually increased in salt content due to lateral infiltration of seawater and groundwater salinity, and the low-salt soil area has gradually increased, affecting the yield of related crops such as the cabbage rapeseed industry.

[0004] Therefore, it is necessary to discover more salt-tolerant genes to improve the salt tolerance of Brassica napus under salt stress conditions. Summary of the invention

[0005] The present invention aims to provide a kind of Brassica napus BnaC05.bZIP68 Application of genes in improving salt tolerance in plants.

[0006] The present invention cloned the Brassica napus gene BnaC05.bZIP68 (BnaC05g24270D), and its related proteins were found to play an important role in improving plant salt tolerance.

[0007] To achieve the present invention, in a first aspect, the present invention provides the use of Brassica napus BnaC05.bZIP68 protein in improving plant salt tolerance.

[0008] In the present invention, the amino acid sequence of the BnaC05.bZIP68 protein is at least one of the following: (1) As shown in SEQ ID NO.2.

[0009] (2) 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] (3) 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.

[0011] (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).

[0012] In a second aspect, the present invention provides Brassica napus BnaC05.bZIP68 Application of genes in improving plant salt tolerance, the BnaC05.bZIP68 The nucleotide sequence of the gene is at least one of the following.

[0013] (1) As shown in SEQ ID NO.1.

[0014] (2) A nucleotide sequence in which one or more nucleotides are replaced, deleted and / or added to the nucleotide sequence shown in SEQ ID NO.1 and the nucleotide sequence expresses a protein with the same function.

[0015] (3) 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.

[0016] (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.

[0017] Plants will highly express the BnaC05.bZIP68 protein in a salt stress environment. At the same time, plants that have been genetically engineered to highly express the above protein are also more adaptable to salt stress environments.

[0018] In a third aspect, the present invention provides a method for improving plant salt tolerance, specifically, increasing the salt tolerance of Brassica napus BnaC05.bZIP68 The expression level of a gene in a plant, and the method thereof includes but is not limited to: (1) Overexpression through genetic engineering BnaC05.bZIP68 Gene vectors are transferred into plants; or

[0019] (2) Select and modify the above mentioned BnaC05.bZIP68 The promoter of the gene is a strong promoter; or

[0020] (3) Increasing the number of chromosomes in plants through genetic engineering BnaC05.bZIP68 the number of copies of a gene; or

[0021] (4) Introducing enhancers through genetic engineering.

[0022] Brassica napus BnaC05.bZIP68 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0023] Preferably, the BnaC05.bZIP68 The gene plasmid is transferred into the plant, and the resulting plant is overexpressed by breeding to homozygosity. BnaC05.bZIP68 In the embodiment of the present invention, the Agrobacterium containing BnaC05.bZIP68 The plasmid of the gene was transformed into Brassica napus to obtain overexpression BnaC05.bZIP68 of genetically modified rapeseed.

[0024] Preferably, the breeding method can be any breeding method that can ultimately obtain homozygotes, including individual selection, pedigree method, haploid breeding, etc., preferably multi-generation breeding.

[0025] In a fourth aspect, the present invention provides a method for increasing the expression level of BnaC05.bZIP68 protein in plants, the method comprising growing the corresponding plants in a salt stress environment, the amino acid sequence of the BnaC05.bZIP68 protein being as shown in SEQ ID NO.2, and the method can increase the expression level of BnaC05.bZIP68 protein in the corresponding plants.

[0026] In a fifth aspect, the present invention provides Brassica napus BnaC05.bZIP68 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 BnaC05.bZIP68 The nucleotide sequence of the gene is shown in SEQ ID NO.1; or

[0027] The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0028] In a sixth aspect, the present invention provides Brassica napus BnaC05.bZIP68 Application of the gene, or the protein encoded by the gene, or the biological material containing the gene, or the method for improving the salt tolerance of plants in plant breeding.

[0029] Brassica napus BnaC05.bZIP68 The nucleotide sequence of the gene is shown in SEQ ID NO.1; or

[0030] The amino acid sequence of the protein is shown in SEQ ID NO.2,

[0031] Preferably, the application in plant breeding is application in cultivating highly salt-tolerant plants or cultivating plants growing in a high-salt soil environment.

[0032] In a seventh aspect, the present invention provides Brassica napus BnaC05.bZIP68 Application of the gene, or the protein encoded by the gene, or the biological material containing the gene, or the method for improving the salt tolerance of plants in the genetic improvement of plant germplasm resources.

[0033] Brassica napus BnaC05.bZIP68 The nucleotide sequence of the gene is shown in SEQ ID NO.1; or

[0034] The protein encoded by it is shown as SEQ ID NO.2.

[0035] Preferably, the plant described in the above uses and / or methods is Brassica napus.

[0036] Beneficial effects of the present invention: The present invention first discovered BnaC05.bZIP68 Gene has the function of improving plant salt tolerance and is overexpressed in plants BnaC05.bZIP68 The gene can significantly improve the growth ability of plants under salt stress and increase the fresh weight of plants. At the same time, growing plants in a salt stress environment can also increase the corresponding plants BnaC05.bZIP68 The expression level of the gene BnaC05.bZIP68 Genes are of great significance in breeding and research related to improving plant salt tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] 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 any creative work.

[0038] Figure 1 The recombinant plasmid constructed on the entry vector pGWC in Example 2 of the present invention pGWC-BnaC05.bZIP68 Schematic diagram of the structure.

[0039] Figure 2 The recombinant plasmid constructed on the plant expression vector pHZM137 in Example 2 of the present invention pHZM137- BnaC05.bZIP68 Schematic diagram of the structure.

[0040] Figure 3Wild type Westar rapeseed under 0.8% NaCl salt stress at different treatment times BnaC05.bZIP68 Schematic diagram of expression levels.

[0041] Figure 4 Wild-type rapeseed and overexpressing BnaC05.bZIP68 Schematic diagram of the growth status and total fresh weight of transgenic rapeseed with the gene. Note: WT in Figure A is wild-type rapeseed, OE is overexpressed BnaC05.bZIP68 Gene-transgenic rapeseed; CK in Figure B is the wild-type rapeseed control, and OE is the overexpression BnaC05.bZIP68 Genetically modified rapeseed. DETAILED DESCRIPTION

[0042] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0043] If no specific techniques or conditions are specified in the examples, all are carried out according to conventional methods or the techniques or conditions described in the literature in the field, or according to the product instructions. If no manufacturer is specified for the reagents and instruments used, all are conventional products that can be purchased through regular channels.

[0044] In the following embodiments.

[0045] Example 1 BnaC05.bZIP68 Gene cloning

[0046] 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 premix (GenStar, A230) to obtain cDNA. BnaC05.bZIP68 CDS sequence, design primer sequence with adapter and stop codon (Forward: SEQ ID NO.3, Reverse: SEQ ID NO.4) to clone BnaC05.bZIP68 The CDS sequence was amplified by PCR using KOD high-fidelity enzyme and sequenced. The gene sequence is shown in SEQ ID NO: 1 and the protein sequence is shown in SEQ ID NO: 2.

[0047] Example 2 BnaC05.bZIP68 Construction of overexpression vector and transformation of rapeseed

[0048] Using the In-Fusion system, the PCR product was connected to the entry vector pGWC digested with AhdI and named pGWC-BnaC05.bZIP68 ( Figure 1 ), positive clones were screened by PCR identification. Next, the Gateway system was used to construct it into the plant expression vector pHZM137 and named pHZM137-BnaC05.bZIP68 ( Figure 2 ), the correctly sequenced plasmid was transferred into GV3101 Agrobacterium to transform Brassica napus Westar (Genome-Wide characterization of DGATsand their expression diversity analysis in response to abiotic stressesin Brassica napus . Yin, X., et al. 22 April 2022 Plants (Basel) 11(9): 1156.), and then multiply the seeds to homozygous and collect them for future use.

[0049] The specific steps for transforming rapeseed 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 a culture room at 22℃ without light for 6-7 days.

[0050] (2) Infection: Plasmids pHZM137-BnaC05.bZIP68 After activation of Agrobacterium, shake the bacteria, collect the cells by centrifugation at 5000 r / min, suspend them in DM and pour them into an empty culture dish for later use; cut the hypocotyls of the seedlings with sterile tweezers and a scalpel, control the length of each hypocotyl to 0.8 - 1.0 cm, place the cut explants in a dish containing the above-mentioned Agrobacterium solution, and immerse them for 10 min; dry the infected explants with sterile filter paper, transfer them to M1 culture medium, and culture them at 22℃ in the dark for 48 h.

[0051] (3) Callus induction: The hypocotyl explants after co-cultivation were transferred to M2 medium for callus induction and cultured under light conditions for 20 days.

[0052] (4) Bud regeneration: Transfer the explants with normal growth and swelling at both ends to differentiation medium M3 and culture them under light conditions. Subculture every 20 days until green buds appear.

[0053] (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.

[0054] The selection 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 selection and verified by PCR.

[0055] Example 3 Wild-type rapeseed under salt stress BnaC05.bZIP68 Changes in gene expression levels

[0056] Wild-type Westar rapeseed was cultured under normal conditions for 5 days, and then treated with 0.8% NaCl solution for 0, 6, 12, and 24 hours. BnaC05.bZIP68 Gene expression level.

[0057] Fluorescence quantitative PCR was performed using 2× RealStar Fast SYBR qPCR Mix (GenStar, A301).

[0058] 1. System: 2×RealStar Fast SYBR qPCR Mix 10 μL, upstream and downstream primers 1 μL each, cDNA 1 μL, ddH2O to 20 μL.

[0059] 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.

[0060] 3. Data analysis used the double ΔCt method.

[0061] The results are as follows Figure 3 As shown, after 6, 12 and 24 hours of salt stress treatment, BnaC05.bZIP68 The expression level of the gene showed a significant upward trend compared with the initial time (0 hours). This phenomenon shows that salt stress can significantly promote BnaC05.bZIP68 The transcriptional activity of the gene was determined, thereby exerting its potential role in coping with salt stress in Brassica napus.

[0062] Example 4 Overexpression BnaC05.bZIP68 Analysis of salt tolerance of transgenic rapeseed

[0063] 1. 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 pot mouth. Put tin foil at the bottom of the pot in advance.

[0064] 2. Choose the ones that are uniform in size and full BnaC05.bZIP68Overexpression rapeseed seeds and wild-type Westar rapeseed seeds were sown into the soil. Three overexpression rapeseed lines were selected, and 6 seeds were planted in each line at a depth of about 2 cm.

[0065] 3. Prepare 0.8% NaCl solution and irrigate each pot with 800 mL for the first time to ensure that water does not leak out. Later, irrigate with salt water once every 5 days, 100 mL each time. The time and amount of salt water can be adjusted according to the humidity.

[0066] 4. After 21 days of salt stress treatment, the total fresh weight (TFW) of rapeseed seedlings was measured.

[0067] like Figure 4 As shown, after 21 days of 0.8% NaCl stress, the overexpressing BnaC05.bZIP68 The fresh weight of rapeseed plants with the gene was significantly higher than that of the wild type, increasing by about 17.8% (Table 1). This indicates that under salt stress conditions, BnaC05.bZIP68 It can improve the salt tolerance of Brassica napus.

[0068]

[0069] The present invention successfully verified the BnaC05.bZIP68 Biological functions of genes in response to salt stress.

[0070] 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 BnaC05.bZIP68 protein in improving plant salt tolerance, characterized in that: The amino acid sequence of the BnaC05.bZIP68 protein is at least one of the following: (1) as shown in SEQ ID NO.2; (2) a protein derived from 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 and retaining the function of the amino acid sequence shown in SEQ ID NO. 2; (3) 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; (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 BnaC05.bZIP68 The application of a gene in improving the salt tolerance of a plant is characterized in that: Said BnaC05.bZIP68 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 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 function; (3) 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; (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 BnaC05.bZIP68 The expression level of a gene in a plant, and the method thereof includes but is not limited to: (1) Overexpression through genetic engineering BnaC05.bZIP68 The gene vector is transferred into the plant; or (2) Selection and modification through genetic engineering BnaC05.bZIP68 The promoter of gene expression is a strong promoter; or (3) Increase the amount of coding in plant chromosomes through genetic engineering BnaC05.bZIP68 The copy number of the gene; or (4) Introducing enhancers through genetic engineering; Said BnaC05.bZIP68 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 BnaC05.bZIP68 The gene plasmid is transformed into plants and the seeds are propagated until they are homozygous.

5. A method for increasing the expression of BnaC05.bZIP68 protein in plants, characterized in that: The corresponding plants are grown in a salt stress environment. The amino acid sequence of the BnaC05.bZIP68 protein is shown in SEQ ID NO.

2.

6. Brassica napus BnaC05.bZIP68 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 BnaC05.bZIP68 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.

7. Brassica napus BnaC05.bZIP68 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 BnaC05.bZIP68 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.

8. The use according to claim 7, characterized in that: The application in plant breeding is to cultivate plants with high salt tolerance or plants growing in a high-salt soil environment; Brassica napus BnaC05.bZIP68 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.

9. A Brassica napus L. BnaC05.bZIP68 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 BnaC05.bZIP68 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.

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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