Application of Brassica napus BnaC05.bZIP68 gene in improving plant salt tolerance

Abstract

The present invention relates to the field of agricultural biotechnology, and in particular to a Brassica napus BnaC05.bZIP68 The present invention provides an application of a gene in improving plant salt tolerance. BnaC05.bZIP68 The gene, whose sequence is shown in SEQ ID NO.1, and the protein sequence encoded by it is shown in SEQ ID NO.2. The present invention found that wild-type Brassica napus is highly expressed in a salt stress environment. BnaC05.bZIP68 gene, and the transgenic Brassica napus overexpressing this gene had stronger salt tolerance, which verified that BnaC05.bZIP68 The gene plays a role as a positive regulatory factor in enhancing the salt tolerance of rapeseed, and provides a key molecular tool for the development of new varieties of Brassica napus with high salt tolerance. This gene has broad application potential and market value in the field of agricultural cultivation, 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 plant salt tolerance. Background Art

[0002] Soil salinization is a growing global problem that hinders plant growth and crop yields. A growing 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 Brassica napus L. is one of the three major types of oilseed rape. It is a composite species that evolved from amphidiploidization following natural interspecific hybridization between Brassica rapa (AA, n = 10) and Brassica oleracea (CC, n = 9). Native to Europe, it is named for its leaf and plant shape resembling that of Brassica oleracea. Its chromosome set is AACC, with an n = 19 chromosomes. Brassica napus has the highest grain yield of the three oilseed rape varieties (Brassica rapa, Brassica juncea, and Brassica napus). It is widely cultivated in the middle and lower reaches of the Yangtze River in my country and is a major oilseed crop. However, soil salinity in some areas of the middle and lower reaches of the Yangtze River has been increasing due to lateral infiltration of seawater and groundwater salinization, resulting in an increase in low-salinity soil areas, impacting the yield of the Brassica napus industry and related crops.

[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 purpose of the present invention is to provide a kind of Brassica napus BnaC05.bZIP68 Application of genes in improving plant salt tolerance.

[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:

[0009] (1) As shown in SEQ ID NO.2.

[0010] (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.

[0011] (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.

[0012] (4) An amino acid sequence obtained by connecting a tag, an enzyme cleavage site and / or a connecting peptide sequence to the N-terminus and / or C-terminus of any of the amino acid sequences (1) to (3).

[0013] 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.

[0014] (1) As shown in SEQ ID NO.1.

[0015] (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 functional properties.

[0016] (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 0.1× SSPE containing 0.1% SDS or 0.1× SSC containing 0.1% SDS at 65°C and the membrane is washed with the solution.

[0017] (4) A nucleotide sequence that has more than 90% homology with the nucleotide sequence shown in SEQ ID NO.1 and expresses the same functional protein.

[0018] 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.

[0019] 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 may be determined by methods including but not limited to:

[0020] (1) Overexpression through genetic engineering BnaC05.bZIP68 Gene vectors are transferred into plants; or

[0021] (2) Select and transform the above-mentioned BnaC05.bZIP68 The promoter of the gene is a strong promoter; or

[0022] (3) Increase the number of plant chromosomes through genetic engineering BnaC05.bZIP68 the copy number of the gene; or

[0023] (4) Introducing enhancers through genetic engineering.

[0024] Brassica napus BnaC05.bZIP68 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0025] 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 an 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.

[0026] 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.

[0027] 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, wherein the amino acid sequence of the BnaC05.bZIP68 protein is shown in SEQ ID NO. 2, and the method can increase the expression level of the BnaC05.bZIP68 protein in the corresponding plants.

[0028] In a fifth aspect, the present invention provides Brassica napus BnaC05.bZIP68 Use of a gene, or a protein encoded by the gene, or a biological material containing the gene, or the above-mentioned method for improving plant salt tolerance in increasing the fresh weight of plants under salt stress;

[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] 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 plant salt tolerance in plant breeding.

[0032] Brassica napus BnaC05.bZIP68 The nucleotide sequence of the gene is shown in SEQ ID NO.1; or

[0033] The amino acid sequence of the protein is shown in SEQ ID NO.2,

[0034] Preferably, the application in plant breeding is application in cultivating highly salt-tolerant plants or cultivating plants growing in a high-salt soil environment.

[0035] 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 above-mentioned method for improving plant salt tolerance in genetic improvement of plant germplasm resources.

[0036] Brassica napus BnaC05.bZIP68 The nucleotide sequence of the gene is shown in SEQ ID NO.1; or

[0037] The protein encoded by it is shown in SEQ ID NO.2.

[0038] Preferably, the plant described in the above uses and / or methods is Brassica napus.

[0039] Beneficial effects of the present invention:

[0040] The present invention first discovered BnaC05.bZIP68 The 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 environment and increase the fresh weight of plants. At the same time, growing plants in salt stress environment can also increase the BnaC05.bZIP68 Gene expression level, indicating BnaC05.bZIP68 Genes are of great significance in breeding and research related to improving plant salt tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to 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.

[0042] 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.

[0043] 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.

[0044] Figure 3 Wild type Westar rapeseed under 0.8% NaCl salt stress at different treatment times BnaC05.bZIP68 Schematic diagram of expression levels.

[0045] Figure 4 wild-type rapeseed and overexpressing BnaC05.bZIP68 Schematic diagram of the growth status and total fresh weight of transgenic rapeseed. 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

[0046] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0047] Where specific techniques or conditions are not specified in the examples, all methods were performed according to conventional methods, techniques or conditions described in literature in the field, or according to product specifications. Reagents and instruments used, for which the manufacturers are not specified, are conventional products that can be purchased through regular channels.

[0048] In the following embodiments.

[0049] Example 1 BnaC05.bZIP68 Gene cloning

[0050] Leaf RNA was extracted using the Tiangen Biochemical Co., Ltd. RNA Extraction Kit (Tiangen, DP432) 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, designed primer sequence with adapter and without stop codon (Forward: SEQ ID NO.3, Reverse: SEQ ID NO.4) for cloning 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.

[0051] Example 2 BnaC05.bZIP68 Construction of overexpression vector and transformation of rapeseed

[0052] The PCR product was connected to the entry vector pGWC digested with AhdI using the In-Fusion system and named pGWC-BnaC05.bZIP68 ( Figure 1 ), positive clones were screened by PCR identification. Next, the clone was constructed into the plant expression vector pHZM137 through the Gateway system 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 the seeds were propagated to homozygous and then harvested for future use.

[0053] The specific steps for transforming rapeseed are as follows:

[0054] (1) Disinfection: Soak plump rapeseed Westar seeds in 75% alcohol for 1 min, disinfect with 0.15% mercuric chloride solution for 12 min, wash with sterile ddH2O 5-6 times, sow on M0 culture medium, and culture in a culture room at 22℃ under dark conditions for 6-7 days.

[0055] (2) Infection: Plasmid-containing pHZM137-BnaC05.bZIP68 After activation of Agrobacterium, shake the culture, collect the cells by centrifugation at 5000 r / min, suspend them in DM suspension and pour them into an empty culture dish for use; use sterile tweezers and a scalpel to cut the hypocotyls of the seedlings, controlling 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 medium, and culture them at 22°C in the dark for 48 h.

[0056] (3) Callus induction: The hypocotyl explants after co-culture were transferred to M2 medium for callus induction and cultured under light conditions for 20 days.

[0057] (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 once every 20 days until green buds appear.

[0058] (5) Rooting: The differentiated green buds 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.

[0059] Hygromycin B (Hyg, Roche, 200 μl / L), a selective antibiotic, 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.

[0060] Example 3 Wild-type rapeseed under salt stress BnaC05.bZIP68 Changes in gene expression levels

[0061] Wild-type Westar rapeseed was cultured under normal conditions for 5 days, and then 0.8% NaCl solution was added for 0, 6, 12 and 24 hours. BnaC05.bZIP68 Gene expression level.

[0062] Fluorescence quantitative PCR was performed using 2× RealStar Fast SYBR qPCR Mix (GenStar, A301).

[0063] 1. System: 2× RealStar Fast SYBR qPCR Mix 10 μL, upstream and downstream primers 1 μL each, cDNA 1 μL, ddH2O to 20 μL.

[0064] 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.

[0065] 3. Data analysis used the double ΔCt method.

[0066] 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 to the initial time (0 hours). This phenomenon shows that salt stress can significantly promote BnaC05.bZIP68 The transcriptional activity of the gene was detected, thereby exerting its potential role in coping with salt stress in Brassica napus.

[0067] Example 4 Overexpression BnaC05.bZIP68 Analysis of salt tolerance of transgenic rapeseed

[0068] 1. Fill a small red pot with a 17 cm diameter soil mixture (vermiculite:nutrient soil = 1:1) until the pot is level with the rim. Place tin foil at the bottom of the pot.

[0069] 2. Choose the ones that are uniform in size and full BnaC05.bZIP68Overexpressing rapeseed seeds and wild-type Westar rapeseed seeds were sown in the soil. Three overexpressing rapeseed lines were selected, and six seeds were planted in each line at a depth of approximately 2 cm.

[0070] 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.

[0071] 4. After 21 days of salt stress treatment, the total fresh weight (TFW) of rapeseed seedlings was measured.

[0072] 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.

[0073]

[0074] The present invention has successfully verified the BnaC05.bZIP68 Biological functions of genes in response to salt stress.

[0075] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

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) an amino acid sequence obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence in (1); The plant is Brassica napus.

2. Brassica napus BnaC05.bZIP68 The application of a gene in improving plant salt tolerance is characterized in that: described BnaC05.bZIP68 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the plant is Brassica napus.

3. A method for improving plant salt tolerance, characterized in that: Increase BnaC05.bZIP68 The expression level of a gene in a plant can be determined by one of the following methods: (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 Gene copy number; or (4) Introducing enhancers through genetic engineering; described BnaC05.bZIP68 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The plant is Brassica napus.

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 for generations until they are homozygous.

5. Brassica napus BnaC05.bZIP68 Use of a gene, or a protein encoded by the gene, or a biological material containing the gene, or the method according to claim 3 or 4, 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; The plant is Brassica napus.

6. Brassica napus BnaC05.bZIP68 Use of a gene, or a protein encoded by the gene, or a biological material containing the gene, or the method according to claim 3 or 4 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; The application in plant breeding is to cultivate plants with high salt tolerance; The plant is Brassica napus.

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 genetic improvement of plant germplasm resources; The genetic improvement of plant germplasm resources is to improve the growth ability of plants under salt stress environment and increase the fresh weight of plants; 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; The plant is Brassica napus.

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