Application of BnaC04.bZIP16 gene in improving plant salt tolerance

Abstract

This invention relates to the field of agricultural biotechnology, specifically to... BnaC04.bZIP16 Application of genes in improving plant salt tolerance. This invention provides... BnaC04.bZIP16 The gene, whose sequence is shown in SEQ ID NO.1, and whose encoded protein sequence is shown in SEQ ID NO.2, is found in this invention to be highly expressed in wild-type Brassica napus after irrigation with salt water. BnaC04.bZIP16 Genes, and transgenic rapeseed overexpressing this gene, can grow better under salt stress conditions; knockout gene... BnaC04.bZIP16 The genetically modified rapeseed is more difficult to grow in salt-stressed environments, thus verifying... BnaC04.bZIP16 This gene plays a positive regulatory role in enhancing the salt tolerance of rapeseed and provides a key molecular tool for developing new rapeseed varieties with high salt tolerance. This gene has broad application potential and commercial value in the field of agricultural planting, especially in improving the salt tolerance of crops and breeding highly salt-tolerant transgenic crops.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more particularly to rapeseed (Brassica napus). BnaC04.bZIP16 Application of genes in enhancing plant tolerance to salt stress. Background Technology

[0002] With global climate change and inadequate land management practices, soil salinization has become one of the major abiotic stresses restricting agricultural production and crop growth. Besides measures such as proper irrigation and drainage and soil improvement, actively developing salt-tolerant crops through genetic engineering is also an effective approach.

[0003] As an important oilseed crop in China, rapeseed (Brassica napus) is significantly affected by soil salinization. Soil salinization not only limits the growth of rapeseed but also affects its yield and quality. In the middle and lower reaches of the Yangtze River, a major rapeseed producing area in my country, the soil salinity is gradually increasing due to seawater infiltration and groundwater salinization, leading to a growing area of ​​low-salt soil and impacting the yield of rapeseed and other related crops. Therefore, cultivating rapeseed varieties suitable for growth in saline soil environments and those that can tolerate salt stress is of paramount importance. Summary of the Invention

[0004] The purpose of this invention is to provide a BnaC04.bZIP16 Application of genes in improving plant salt tolerance.

[0005] This invention clones the gene of Brassica napus. BnaC04.bZIP16 (BnaC04g09600D), and found that it plays an important role in improving the salt tolerance of plants.

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

[0007] In this invention, the amino acid sequence of the BnaC04.bZIP16 protein is at least one of the following:

[0008] (1) As shown in SEQ ID NO.2.

[0009] (2) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in SEQ ID NO.2 and has the same function as a protein.

[0010] (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 substituting, deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.2.

[0011] (4) An amino acid sequence obtained by attaching a tag, restriction site and / or linking a peptide sequence to the N-terminus and / or C-terminus of any of the amino acid sequences in (1)-(3).

[0012] Secondly, the present invention provides a type of rapeseed. BnaC04.bZIP16 The application of genes in improving plant salt tolerance, characterized in that, the BnaC04.bZIP16 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 that hybridizes with the sequence shown in SEQ ID NO.1 under stringent conditions and expresses the same functional protein, wherein the stringent conditions are hybridization at 65°C in 0.1 × SSPE containing 0.1% SDS or 0.1 × SSC containing 0.1% SDS and washing the membrane with the solution.

[0015] (3) A nucleotide sequence that expresses the same functional protein by substituting, deleting and / or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID NO.1.

[0016] (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.

[0017] This invention discovers that plants highly express [certain substances] under salt stress. BnaC04.bZIP16 The protein edited by the gene, and plants that express the protein highly through genetic engineering, are better adapted to salt stress environments, while plants that have the gene knocked out are more susceptible to the effects of salt stress, resulting in reduced yields.

[0018] Thirdly, the present invention provides a method for improving the salt tolerance of plants, specifically by increasing the aforementioned rapeseed gene. BnaC04.bZIP16 The expression level of the encoded protein in the plant, and the methods include, but are not limited to, the following.

[0019] (1) Using genetic engineering techniques to overexpress BnaC04.bZIP16 Gene vectors are transferred into plants; or

[0020] (2) Selecting and modifying the aforementioned through genetic engineering techniques BnaC04.bZIP16 The gene promoter is a strong promoter; or

[0021] (3) Introducing enhancers through genetic engineering; or

[0022] (4) Increasing the number of chromosomes in plants through genetic engineering. BnaC04.bZIP16 Gene copy number.

[0023] In some embodiments, by including BnaC04.bZIP16 The method of transforming gene plasmids into plants and breeding them to homozygosity allows the resulting plants to overexpress the gene. BnaC04.bZIP16 Genes, thereby acquiring higher salt tolerance. In this invention, Agrobacterium is used to... BnaC04.bZIP16 The gene plasmid was transformed into Brassica napus and overexpressed. BnaC04.bZIP16 Genetically modified rapeseed.

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

[0025] Fourthly, the present invention provides a method for increasing the expression level of BnaC04.bZIP16 protein in plants, specifically by growing the corresponding plants in a salt stress environment. The amino acid sequence of the BnaC04.bZIP16 protein is shown in SEQ ID NO.2. Growing in a salt stress environment can increase the expression level of BnaC04.bZIP16 protein in plants.

[0026] Fifthly, the present invention provides rapeseed of the Brassica oleracea type. BnaC04.bZIP16 The application of genes, or the proteins they encode, or biological materials containing such genes, or the aforementioned methods for improving plant salt tolerance in plant breeding;

[0027] The 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, viral vector, engineered bacteria, or non-renewable plant cells or tissues.

[0030] Preferably, the application in plant breeding is the application in cultivating highly salt-tolerant plants or plants that grow in high-salt soil environments.

[0031] Sixthly, the present invention provides rapeseed of the Brassica oleracea type. BnaC04.bZIP16 The application of genes, or the proteins they encode, or biological materials containing such genes, or the aforementioned methods for improving plant salt tolerance in increasing the fresh weight of plants under salt stress.

[0032] The Brassica napus BnaC04.bZIP16 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] The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria, or non-renewable plant cells or tissues.

[0035] Seventhly, the present invention provides rapeseed of the Brassica oleracea type. BnaC04.bZIP16 The application of genes, or the proteins they encode, or biological materials containing such genes, or the aforementioned methods for improving plant salt tolerance in the genetic improvement of plant germplasm resources;

[0036] The Brassica napus BnaC04.bZIP16 The nucleotide sequence of the gene is shown in SEQ ID NO.1; or

[0037] The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0038] The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria, or non-renewable plant cells or tissues.

[0039] The beneficial effects of this invention are:

[0040] This invention is the first discovery BnaC04.bZIP16 Genes that enhance salt tolerance in plants are overexpressed. BnaC04.bZIP16 The gene can significantly improve a plant's growth ability under salt stress, while plants with the gene knocked out are more difficult to grow under salt stress, indicating that... BnaC04.bZIP16 Genes play an important role in breeding programs that improve plant salt tolerance. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0042] Figure 1 In Embodiment 2 of the present invention, the In-Fusion system is used to... BnaC04.bZIP16 The CDS sequence was constructed from the recombinant plasmid on the entry vector pGWC. pGWC-BnaC04.bZIP16 A structural diagram.

[0043] Figure 2 In Embodiment 2 of the present invention, the Gateway system is used to connect the entry carrier... BnaC04.bZIP16 Recombinant plasmid constructed on the plant expression vector pHZM137 pHZM137-BnaC04.bZIP16A structural diagram.

[0044] Figure 3 As in Embodiment 2 of the present invention Bsa I. Restriction endonucleases and T4 ligases were used to construct a gene editing vector containing the target sequence from the PCR product into the pHSE401 vector. pHSE401-giBnbZIP16 Structural diagram.

[0045] Figure 4 Wild-type Westar rapeseed under 0.8% NaCl salt stress for different treatment times BnaC04.bZIP16 The diagram illustrates the expression levels, with error bars representing SD.

[0046] Figure 5 Wild-type rapeseed grown under 0.8% NaCl salt stress for 21 days, overexpression BnaC04.bZIP16 Genetically modified rapeseed and those with knocked-out genes BnaC04.bZIP16 A schematic diagram showing the growth status and total fresh weight of rapeseed in Figure A. Note: In Figure A, WT represents wild-type rapeseed, and OE represents overexpression. BnaC04.bZIP16 Genetically modified rapeseed with knocked-out genes, KO means that the genetic code has been removed. BnaC04.bZIP16 The gene in rapeseed; in Figure B, CK represents wild-type rapeseed (control), and OE represents overexpression. BnaC04.bZIP16 Genetically modified rapeseed with knocked-out genes, KO means that the genetic code has been removed. BnaC04.bZIP16 Rapeseed with genetic traits. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0048] Example 1 BnaC04.bZIP16 Cloning of genes

[0049] RNA was extracted from leaves using a Tiangen Biotech RNA extraction kit (Tiangen, DP432), and reverse transcription was performed using an All-in-one RT Mix with gDNA Remover StarScript III premix for genomic de-transcription (GenStar, A230). Follow the steps below.

[0050] RNA extraction:

[0051] 1. Fresh rapeseed leaves were quick-frozen and ground with liquid nitrogen, and 500 μL of SL (495 μL SL, 5 μL β-mercaptoethanol) was added. The mixture was shaken and centrifuged at 1,2000 rpm for 2 min at 4℃. 400 μL of the supernatant was then transferred to a CS column.

[0052] 2. Centrifuge at 1,2000 rpm for 2 min at 4 °C, and transfer the supernatant to a new RNase-Free centrifuge tube. Add 180 μL of anhydrous ethanol, mix well, transfer to CR3, and centrifuge at 12,000 rpm for 30 sec at 4 °C.

[0053] 3. Add 80 μL of DNase I working solution (10 μL) DNase I. Stock solution, 70 μL RDD), let stand at room temperature for 10 min.

[0054] 4. Add 350 μL of protein removal buffer RW1 and 500 μL of wash buffer RW, centrifuge at 12,000 rpm for 1 min. Transfer the CR3 column to a new RNase-Free centrifuge tube, add 30 μL of RNase-Free ddH2O, incubate at room temperature for 2 min, centrifuge to obtain the RNA solution.

[0055] Reverse transcription:

[0056] 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 a final volume of 20 μL.

[0057] 2. After mixing, reverse transcription was performed using a PCR instrument to obtain cDNA. PCR program: 37℃ for 2 min, 50℃ for 15 min, 85℃ for 2 min.

[0058] Gene cloning:

[0059] Searching for sequences from the rapeseed genome database (https: / / yanglab.hzau.edu.cn / BnTIR). BnaC04.bZIP16 The CDS sequence was obtained, and then primer sequences with adapters and no stop codons were designed for cloning (Forward: SEQ ID NO. 3, Reverse: SEQ ID NO. 4). BnaC04.bZIP16 The CDS sequence was obtained, and PCR amplification was performed using KOD high-fidelity enzyme. The sequence was then sequenced and identified. The gene sequence is shown in SEQ ID NO. 1, and the protein sequence is shown in SEQ ID NO. 2.

[0060] Example 2 BnaC04.bZIP16 Construction of overexpression and knockout vectors and transformation of rapeseed

[0061] Overexpression vector:

[0062] Using the In-Fusion system, PCR products were ligated into... Ahd The enzyme was digested in the entry vector pGWC and named... pGWC-BnaC04.bZIP16 ( Figure 1 The *E. coli* DH5α was transformed, and positive clones were screened by PCR. Then, using the Gateway system, these clones were constructed into the plant expression vector pHZM137 and named... pHZM137 - BnaC04.bZIP16 ( Figure 2 The correctly sequenced plasmid was transformed into Agrobacterium GV3101 and transformed into Brassica napus Westar (Genome-Wide characterization 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 collect seeds for future use after breeding to achieve purity.

[0063] Knockout vector construction:

[0064] Search the CRISPR-GE website (http: / / skl.scau.edu.cn / home / ). BnaC04.bZIP16 The gene and its homologs BnaA05g08520D, BnaA03g56410D, and BnaA04g20520D were used to design a common gene editing target site. Primers (Forward: SEQ ID NO.5, Reverse: SEQ ID NO.6) were designed and constructed into a gene editing target site using T4 ligase. Bsa I. Enzyme digestion pHSE401The vector (A CRISPR / Cas9 toolkit for multiplex genome editing in plants. Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, 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. The correctly sequenced plasmids were then transformed into Agrobacterium GV3101 to transform rapeseed, and after purification, the seeds were harvested for later use.

[0065] pHZM137-BnaC04.bZIP16 and pHSE401-giBnbZIP16 The genetic transformation and screening of positive transgenic lines are carried out through the following steps:

[0066] (1) Disinfection: Plump rapeseed Westar seeds were soaked in 75% alcohol for 1 min, disinfected with 0.15% mercuric chloride solution for 12 min, and washed with sterile ddH2O 5-6 times before being sown on M0 medium and cultured in the dark at 22℃ for 6-7 days.

[0067] (2) Infection: Infecting the plasmid-containing pHZM137-BnaC04.bZIP16 or pHSE401-giBnbZIP16 After activation, Agrobacterium was shaken and centrifuged at 4000 r / min to collect the cells. The cells were then suspended in DM suspension and poured into empty culture dishes for later use. The hypocotyls of the seedlings were cut with sterile forceps and a scalpel, each 0.8-1.0 cm in length. The cut explants were placed in a dish containing bacterial suspension and incubated for 10 min. The infected explants were blotted dry with sterile filter paper and transferred to M1 medium. They were then incubated in the dark at 22°C for 48 h.

[0068] (3) Callus induction: The co-cultured hypocotyl explants were transferred to M2 medium for callus induction and cultured under light for 20 days.

[0069] (4) Bud regeneration: Transfer the explants that are growing normally and have swollen ends to the differentiation medium M3 and culture them under light. Subculture them every 20 days until green buds appear.

[0070] (5) Rooting: The differentiated green shoots are transferred to M4 medium for growth. They are subcultured every 20 days until roots grow. After roots grow, they are transferred to flower pots to keep them moist and harden off. After hardening off, they are transplanted into large pots for cultivation.

[0071] Hygromycin B (Hyg, Roche, 200 μl / L) was added to M2, M3 and M4 culture media for screening. After hardening, the overexpression materials were sprayed with herbicide (Basta, Coolaber, 3 ml / L) for screening. DNA was extracted from leaves of the knockout materials and sequenced to determine whether the knockout was successful.

[0072] Example 3 Wild-type rapeseed under salt stress BnaC04.bZIP16 Changes in gene expression levels

[0073] Wild-type Westar was cultured under normal conditions for 5 days, then treated with a 0.8% NaCl solution for 0, 6, 12, and 24 hours, and the results were measured. BnaC04.bZIP16 Gene expression levels.

[0074] Real-time quantitative PCR was performed using 2 × RealStar Fast SYBR qPCR Mix (GenStar, A301). The experimental procedures were followed according to the manufacturer's instructions.

[0075] 1. System: 10 μL of 2 × RealStar Fast SYBR qPCR Mix, 1 μL each of 10 μM forward and reverse primers, 1 μL of cDNA, and ddH2O to bring the total to 20 μL.

[0076] 2. qPCR program: pre-denaturation at 95℃ for 2 min, denaturation at 95℃ for 10 sec, annealing at 60℃ for 30 sec, amplification for 40 cycles.

[0077] 3. Data analysis uses the double ΔCt method.

[0078] The results are as follows Figure 4 Under salt stress treatment for 6, 12, and 24 hours, BnaC04.bZIP16 Gene expression levels were significantly increased compared to 0 hours, indicating that salt can induce... BnaC04.bZIP16 Gene expression in plants.

[0079] Example 4 BnaC04.bZIP16 Analysis of the influence of genes on salt tolerance in rapeseed

[0080] 1. Fill the pot with soil: Fill a small red pot with a diameter of 17 cm with mixed soil (vermiculite: nutrient soil = 1:1), making sure the soil is level with the rim of the pot.

[0081] 2. Seed sowing: Select seeds that are plump and uniform in size. BnaC04.bZIP16 Transgenic seeds (seeds from overexpressed and knockout plants) and Westar seeds were sown in the soil. Three lines were selected from each transgenic material, and 10 seeds were sown in each line at a depth of about 2 cm. After the cotyledons opened, there were 6 seedlings per pot, and the excess were removed.

[0082] 3. Saltwater irrigation: Prepare a 0.8% NaCl solution. Pour 800 mL into each small red basin for the first time, ensuring that the water in each basin does not leak out. Afterward, irrigate once every five days, each time with 100 mL. Adjust the timing and amount of saltwater irrigation according to the humidity. The salt stress treatment lasts for 21 days.

[0083] 4. Biomass determination: The total fresh weight (TFW) of rapeseed seedlings was determined 21 days after salt stress.

[0084] like Figure 5 As shown, after 21 days of 0.8% NaCl stress, compared with the control Westar rapeseed, the overexpression of [a specific substance] was significantly reduced. BnaC04.bZIP16 The fresh weight of the plants (Table 1) was significantly higher than that of the wild type, increasing by approximately 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.

[0085]

[0086] This invention uses rapeseed (Brassica napus) as an example to demonstrate through experiments that... BnaC04.bZIP16 The function of genes in helping plants grow normally under salt stress.

[0087] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

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

2. Overexpression of Brassica napus BnaC04.bZIP16 The application of genes in improving plant salt tolerance is characterized by, The BnaC04.bZIP16 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The plant in question is Brassica napus.

3. A method for improving the salt tolerance of plants, characterized in that, Increase BnaC04.bZIP16 The methods for measuring gene expression levels in plants include, but are not limited to: (1) Using genetic engineering techniques to overexpress BnaC04.bZIP16 Gene vectors are transferred into plants; or (2) Increasing the coding in plant chromosomes through genetic engineering. BnaC04.bZIP16 The copy number of a gene; or (3) Introducing enhancers through genetic engineering; or (4) Selecting and modifying regulation through genetic engineering techniques BnaC04.bZIP16 The promoter for gene expression is a strong promoter; The BnaC04.bZIP16 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The plant in question is Brassica napus.

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

5. A type of rapeseed (Brassica napus) BnaC04.bZIP16 The application of a gene, or the protein it encodes, or biological material containing the gene, or the method of claim 3 or 4 in plant breeding; The 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 materials include recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, and engineered bacteria. The application in plant breeding is to cultivate highly salt-tolerant plants; The plant in question is Brassica napus.

6. A type of rapeseed (Brassica napus) BnaC04.bZIP16 The application of a gene, or the protein it encodes, or biological material containing the gene, or the method of claim 3 or 4 in improving the fresh weight of plants under salt stress. The 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 materials include recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, and engineered bacteria. The plant in question is Brassica napus.

7. A type of rapeseed (Brassica napus) BnaC04.bZIP16 The application of a gene, or the protein it encodes, or biological material containing the gene, or the method described in claim 3 or 4, in the genetic improvement of plant germplasm resources; The 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 materials include recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, and engineered bacteria. The plant in question is Brassica napus; The genetic improvement of plant germplasm resources refers to the improvement of plants' ability to tolerate salt stress.

Images