Application of brassica napus BnaCP15A gene coded protein in improvement of plant salt tolerance

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of a brassica napus BnaCP15A gene coded protein to improvement of plant salt tolerance. The technical problem to be solved by the invention is to provide a new choice for improving the salt tolerance of plants. The technical scheme of the invention is the application of the protein coded by the brassica napus BnaCP15A gene in improving the salt tolerance of the plant, wherein the amino acid sequence of the protein is shown as SEQ ID NO.2. The invention further discloses the application of the protein coded by the brassica napus BnaCP15A gene. The salt tolerance of the plant is improved by increasing the expression of the brassica napus BnaCP15A gene in the plant, and the nucleotide sequence of the BnaCP15A gene is as shown in SEQ ID NO. 1. The main purpose of the invention is to provide the application of the protein coded by the brassica napus BnaCP15A gene in improving the salt tolerance of the plant, a new choice is provided for improving the salt tolerance of the brassica napus, and the brassica napus BnaCP15A gene has important application value.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of a protein encoded by a BnaCP15A gene of Brassica napus in improving plant salt tolerance. Background Art

[0002] Rapeseed is a major oilseed crop worldwide and the fifth-largest crop in my country after rice, wheat, corn, and soybeans. Rapeseed oil production accounts for over 45% of my country's total vegetable oil production. Rapeseed has a taproot system, allowing it to effectively access water, fertilizer, and nutrients from deep soil layers. Compared to fibrous root crops like wheat, corn, and rice in the grass family, rapeseed effectively avoids the stresses of surface salinity and alkali soils, providing technical support for the development of saline-alkali land. Previous research has found that rapeseed boasts high biological yields, a wide range of suitable planting areas, and strong salt-alkali tolerance. It has enormous potential for application in the restoration, improvement, and utilization of saline-alkali land, making it a pioneering crop in the improvement and comprehensive development of saline-alkali land. By balancing grain and oil production with soil improvement, it offers the advantage of both improving and generating profits. This also signals a shift in saline-alkali land development from primarily renovating and adapting crops to more appropriate crops, significantly impacting rapeseed supply security, soil remediation, and ecological conservation.

[0003] However, despite its salt-alkali tolerance, rapeseed's growth and development are significantly inhibited in soils with high salinity and alkali content, making it difficult to guarantee yield and quality. Salt stress is a key abiotic stressor in agricultural production, severely impacting plant growth and development throughout its life cycle, hindering sustainable agricultural development and ecological stability. Therefore, improving rapeseed's salt tolerance is crucial for ensuring yield and quality and promoting the effective utilization of saline-alkali land.

[0004] Plant cysteine ​​proteases (CPs), also known as thiol proteases, are an important family of hydrolytic proteases in plants, primarily localized in the vacuole, cytoplasm, and cell wall. CPs play important roles in programmed cell death, seed development, organ senescence, protein mobilization, and responses to biotic and abiotic stresses. Studies have reported that CPs participate in various stress responses, including those to high temperature, drought, and salinity. For example, the Arabidopsis cysteine ​​protease gene RD19 is strongly induced to express under high salt and osmotic stress conditions; when the sweet potato SPCP2 gene is overexpressed in Arabidopsis, the plants show enhanced resistance to drought and salt stress; the TaCP gene in wheat seedlings is significantly upregulated after treatment with polyethylene glycol (PEG) and sodium chloride (NaCl), and transgenic Arabidopsis plants overexpressing TaCP show stronger drought tolerance and higher CP activity than wild-type Arabidopsis under water-deficient conditions; in pepper, the cysteine ​​protease gene CaCP negatively regulates salt and osmotic stress to induce leaf senescence; in Salix psammophila, overexpression of SmCP can enhance the plant's salt tolerance; apple MdCP37 negatively regulates the resistance of apple callus to salt stress, and overexpression of tobacco lines reduces salt stress resistance; wheat TaCP3 is involved in abiotic stresses such as drought, high salt, low temperature and high temperature.

[0005] Although studies have shown that certain CP genes contribute to salt-alkali tolerance in other plants, there are currently few reports on enhancing salt tolerance in rapeseed by increasing the expression of specific CP genes. The specific functions and mechanisms of action of these specific rapeseed CP genes in rapeseed remain unclear, and their direct application to improving salt tolerance in rapeseed presents certain difficulties and challenges. Therefore, developing an effective method to improve salt tolerance in rapeseed is an urgent issue. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention provides a new option for improving the salt tolerance of rapeseed.

[0007] The technical solution of the present invention is: Application of a protein encoded by the BnaCP15A gene of Brassica napus in improving plant salt tolerance, wherein the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0008] Preferably, the plant is rapeseed.

[0009] Furthermore, the improvement of plant salt tolerance is achieved by increasing the expression level of the BnaCP15A gene in the plant.

[0010] Furthermore, the method for increasing the expression level of the BnaCP15A gene in plants is: constructing a plant expression vector expressing the BnaCP15A gene, and transforming the plant.

[0011] Preferably, the backbone vector of the plant expression vector is pCAMBIA2300.

[0012] Specifically, the transformation is mediated by Agrobacterium.

[0013] Beneficial effects of the present invention: the present invention provides the application of the protein encoded by the BnaCP15A gene of Brassica napus in improving plant salt tolerance. In the process of salt-resistant breeding, the salt tolerance of rape can be significantly improved by increasing the expression of the above-mentioned BnaCP15A gene, and new effective ways and methods are provided for the salt-alkali tolerant variety breeding of rape, with good application prospects. The experiments in the embodiments of the present invention have confirmed that the BnaCP15A gene is transferred into Brassica napus and overexpressed. After salt stress treatment, the fresh weight and dry weight of the overexpressed plant are significantly higher than those of the wild type. The plant transferred into the BnaCP15A gene has a significantly improved biomass at the seedling stage under a salt stress environment. Therefore, the method for overexpressing the BnaCP15A gene of Brassica napus provided by the present invention can improve the salt tolerance of rape, has important application value, and is of great significance to the improvement of the salt-tolerant variety of rape. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Phylogenetic tree of the RD19A gene.

[0015] Figure 2 Salt-induced expression of BnaCP15A gene. Relative expression: relative expression level.

[0016] Figure 3 Electrophoresis diagram of target gene clone, M represents marker, 1, 2, and 3 represent three replicates of target gene fragment clone.

[0017] Figure 4 Diagram of protein tertiary structure.

[0018] Figure 5 Phylogenetic tree diagram.

[0019] Figure 6 Electrophoresis diagram of positive seedling identification. Each lane is the DNA positive identification of a single strain. 1 to 12 are the positive strain numbers, and M is a marker.

[0020] Figure 7 Salt stress phenotype of rapeseed at the seedling stage.

[0021] Figure 8 Fresh weight chart at the seedling stage, Fresh weigh indicates fresh weight.

[0022] Figure 9 Seedling dry weight chart, Dry weight means dry weight. DETAILED DESCRIPTION

[0023] The present invention will be further described in detail below by way of examples, but the present invention is not limited thereto in any way.

[0024] Example 1 Screening of BnaCP15A gene According to literature reports, Arabidopsis RD19A can respond to salt stress, but its molecular function is unknown. In Arabidopsis, there are 29 members of this protein family. Phylogenetic tree analysis of the 29 homologous genes of this gene in Arabidopsis revealed that the RD19D gene has a high homology with the RD19A gene (see Figure 1 ), by comparing the RD19D gene sequence of Arabidopsis thaliana (the RD19D gene sequence of Arabidopsis thaliana can be found through the Arabidopsis database TAIR, accession number AT3G54940) to that of Brassica napus, it was found that its homologous gene in rapeseed is BnaCP15A. At the same time, through the transcriptome data analysis of Brassica napus Huayouza 62 and Zhongshuang 11 after salt stress treatment, it was found that the BnaCP15A gene can be significantly induced by salt (see for details). Figure 2 ), but its salt tolerance function in rapeseed has not yet been determined.

[0025] To further investigate the salt tolerance function of the BnaCP15A gene, a subsequent example of the present invention involved constructing an overexpression vector for BnaCP15A (SEQ ID NO. 1 includes the UTR sequence, but only the ATG to TAG sequence was amplified during the overexpression vector construction, omitting the UTR sequence) and transforming it into Brassica napus Westar to investigate its biological role in salt tolerance. This process involved designing primers, cloning the gene fragment using DNA from Huayouza 62 as a template, constructing an overexpression vector via homologous recombination, and transforming it into the large intestine. Sequencing the result (SEQ ID NO. 7) confirmed the successful construction of the overexpression vector, which was then transformed into Brassica napus Westar via Agrobacterium-mediated transfection. The overexpression vector was then evaluated for salt tolerance in seedlings of plants overexpressing the BnaCP15A gene.

[0026] Example 2 Cloning of the rapeseed BnaCP15A gene 1. Rapeseed DNA Extraction (1) Preheat the CTAB solution in a 65°C water bath for 30 minutes; (2) Take a certain amount of young tissue of Brassica napus Huayouza 62 and place it in a 2 mL EP tube. Add two steel balls and liquid nitrogen, and shake vigorously until the plant tissue becomes powder. (3) Add 500 μL of preheated CTAB, incubate in a 65°C water bath for 30 min, remove, and cool to room temperature; (4) Add an equal volume of chloroform-isoamyl alcohol (volume ratio 24:1) mixture and invert vigorously; (5) Centrifuge at 12,000 rpm for 10 min at 25°C. After centrifugation, separate layers appear. Pipette 300 µL of the top layer into a new EP tube. (6) Add 1000 μL of anhydrous ethanol, mix well, and place in a -20°C refrigerator to precipitate for more than 2 hours; (7) Centrifuge at 12,000 rpm for 10 min and discard the supernatant; (8) Add 900 μL of 75% ethanol, centrifuge at 12,000 rpm for 2 min, and discard the supernatant; (9) Place the centrifuge tube upside down on the filter paper and bake in an oven at 60°C for 5 minutes; (10) Remove from the oven, dissolve the DNA in 100 µL of dd water, and store at -20°C.

[0027] 2. Gene cloning The general idea is to use the extracted DNA as a template and amplify with a high-fidelity enzyme.

[0028] The PCR reaction system (50 μL) is shown in Table 1.

[0029] Table 1 PCR reaction system (50 µL) The PCR reaction program was as follows: 95°C for 5 min; 34 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 2 min; and 72°C for 10 min.

[0030] (1) Searching for the location of the rapeseed BnaCP15A gene in the genome on the BnIR website revealed that rapeseed contains two copies of BnaCP15A, located on chromosomes A09 and C08 respectively; The gDNA sequence (nucleotide sequence) of the rapeseed BnaCP15A gene was searched on the BnIR website (accession number: BnaC08G0298900WE). It was found that the BnaCP15A nucleotide sequence is shown in SEQ ID NO. 1 (SEQ ID NO. 1 is the sequence of the CO8 copy of the BnaCP15A gene): It can be further seen that the amino acid sequence of BnaCP15A is shown in SEQ ID NO.2: MLAKTLALVLTCTAIFCHVAVSLEDLTIRQVTEDRLPNLLGTHTERKFRVFMSDYGKSYSTREEYIHRLGIFAKNVLKAAEHQMMDPTAVHGVTQFSDLTEDEFKRMYNGVVDVDRGHGGVVGAEAPKLEIKGLPKDFDWREKGAVTEVKDQGACGSCWAFSTTGAVEGANFVSTGKLVSLS EQQLVDCDKACDPKDKKACDNGCGGGLMTNAYEYLEKAGGLEEEKSYPYTGKRGHCKFDPKKVAVKIVNFTNIPLDEDQITARLVQHGPLSVGINAVFMQTYIGGVSCPLICSKKRLNHGVLLVGYGSKGFSILRLSNKPYWIIKNSWGKKWGDNGYYKLCRGHDMCGINSMASAVLTQVSS (2) Design specific primers BnaCP15A-F and BnaCP15A-R based on the target gene, add vector homology arms to the 5' end of the primers, clone the BnaCP15A gene by PCR and sequence; the sequences of BnaCP15A-F and BnaCP15A-R are as follows: BnaCP15A-F: 5'-caggtcgactctagaggatccATGTTGGCTAAAACCTTAGCACTGG (SEQ ID NO. 3, lowercase letters represent vector homology arms) BnaCP15A-R: 5'-cgagaattcgagctcggtaccCTATGACGAAACTTGGGTAAGCAC (SEQ ID NO. 4, lowercase letters represent vector homology arms) (3) Take 5µL of the product and perform agarose gel electrophoresis. The test results are as follows: Figure 3 As shown; (4) Recover the product using the Tiangen kit and store it at 4°C.

[0031] The amino acid sequence of BnaCP15A is shown in SEQ ID NO. 2. It can be seen that the BnaCP15A protein contains 364 amino acids. The tertiary structure of the BnaCP15A protein was predicted using the AlphaFold Protein Structure Database. The results are as follows: Figure 4As shown, it contains a signal peptide structure at the N-terminus. According to the NCBI database, the BnaCP15A protein also contains a cathepsin propeptide inhibitor domain (I29) and a C1 family peptidase domain.

[0032] The phylogenetic tree analysis was performed on genes in rapeseed, Arabidopsis, radish, wheat, sweet potato, apple, psammophila, and pepper, such as Figure 5 As shown, in rapeseed, the gene is more closely related to radish and more distantly related to other species.

[0033] Example 3 Construction of BnaCP15A overexpression vector (1) Vector linearization and enzyme digestion: The pCAMBIA2300 vector was linearized by double enzyme digestion (BamHI and KpnⅠ). After completion, the enzyme digested plasmid was purified and recovered using the Tiangen kit. The enzyme digestion system (50µL) is shown in Table 2: Table 2 Enzyme digestion system (50µL) Reaction conditions: 37℃ for 30 min.

[0034] (2) The BnaCP15A gene obtained by PCR amplification in Example 2 was ligated with the pCAMBIA2300 linearized vector by homologous recombination technology to obtain a recombinant plasmid. The BnaCP15A gene is located behind the 35S promoter. Driven by the 35S promoter, BnaCP15A can be overexpressed in plants. The KanR gene expression cassette is assembled on the vector plasmid as a transgenic screening marker, and transgenic plants can be screened with kanamycin. The recombinant system (20 μL) is shown in Table 3: Table 3 Reconstitution system (20µL) Reaction conditions: 37℃ for 30 min.

[0035] (3) To confirm whether the overexpression vector was successfully constructed, PCR reaction and sequencing analysis were performed. The primers were constructed based on the BnaCP15A gene fragment sequence (forward primer) and the pCAMBIA2300 vector (reverse primer). The primer sequences used are as follows: BnaCP15A-F: 5'-ATGTTGGCTAAAACCTTAGCACTGG (SEQ ID NO.5) M13-48R: 5'-AGCGGATAACAATTTCACACAGGA (SEQ ID NO.6) Sequencing analysis was performed at Qingke Biotechnology Co., Ltd., and sequence alignment confirmed that the results were correct. The nucleotide sequence is shown in SEQ ID NO.7: SEQ ID NO. 1 was obtained by querying the BnIR website and includes the UTR sequence, while SEQ ID NO. 7 is the sequencing result and only includes the sequence from ATG to TAG. Therefore, SEQ ID NO. 7 is included in SEQ ID NO. 1. Furthermore, both SEQ ID NO. 7 and SEQ ID NO. 1 are sequences of the CO8 copy of the BnaCP15A gene.

[0036] PCR detection and sequencing verification confirmed that the overexpression vector was successfully constructed and named OE-CP15A.

[0037] Example 4 Obtaining Overexpressed Transgenic Materials 1. Obtaining Agrobacterium containing BnaCP15A gene overexpression vector The overexpression vector OE-CP15A obtained in Example 3 was transformed into Agrobacterium GV3101 competent cells, and positive bacteria were selected for preservation.

[0038] (1) Take out the competent cells (stored at -80℃), wait for them to partially melt at room temperature or in the palm of your hand for a while, and insert them into ice when they are in the state of ice-water mixture.

[0039] (2) Add 5 µL of plasmid DNA to every 30 µL of competent medium, mix thoroughly by pipetting, and place in an ice bath for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and on ice for 5 min.

[0040] (3) Add 200 μL of antibiotic-free LB liquid medium and incubate at 28°C in a shaking incubator for 1-1.5 h.

[0041] (4) Spread the bacterial solution on solid LB medium (containing the corresponding antibiotics) and culture in a 28°C incubator until uniform single colonies grow on the solid medium.

[0042] (5) Pick the spots and identify the positive colonies by PCR for bacterial preservation.

[0043] 2. Genetic transformation of rapeseed hypocotyl The BnaCP15A gene was transformed into Brassica napus Westar using Agrobacterium-mediated method.

[0044] (1) Seed sterilization and sowing: Select seeds with full grains and sterilize them by soaking them in 75% alcohol for 1 minute. Pour off the alcohol and rinse them with sterile water. Sterilize them with 0.1% mercuric chloride for 10-15 minutes and rinse them with sterile water 4-5 times, soaking them for 5 minutes each time. Sow the sterilized seeds on M0 medium and incubate them in the dark at 25°C for 5-6 days.

[0045] (2) Preparation of bacterial solution: 3-4 days after sowing the seeds, take out the transformed strain stored at -80℃, streak it on a double-antibody (gentamicin and kanamycin) LB plate, and culture it at 28℃ for 48h; pick a single colony and inoculate it into 1mL of triple-antibody (rifampicin, gentamicin, and kanamycin) LB medium, and culture it at 28℃ with a shaker (180r / min) for 24h. Take 100μL of bacterial solution and inoculate it into a flask containing 100mL of double-antibody LB medium, and culture it at 28℃ until the OD600 is about 0.5 (about 12-14h); centrifuge it at 4000rpm / min for 10min to collect the bacterial solution, add an equal volume of DM medium (final AS concentration is 0.05mmol) to suspend the bacteria, and culture it at 28℃ with a shaker (180r / min) for 2-3h. Pour it into a culture dish for later use.

[0046] (3) Infection and co-cultivation: Remove the seedlings that have been cultured in the dark for 6 days and cut the hypocotyls using sterilized forceps and a scalpel. Cut as quickly and vertically as possible to ensure a smooth incision of 0.8-1 cm in length. Place the explants in the prepared Agrobacterium solution and infect for 8-10 minutes (not too long). Pour out the solution and spread the explants on sterilized filter paper to absorb any excess solution on the surface. Use forceps to evenly place the explants on the ML medium, ensuring that both ends of the explants are in full contact with the medium. Incubate in the dark for 24-36 hours.

[0047] (4) Selection and callus induction: After co-cultivation, the explants were removed from the dark and transferred to M2 medium for selection and callus induction, containing 100 mg / L kanamycin as a selection antibiotic and timentin to inhibit the growth of Agrobacterium. The explants were cultured at 25°C for 16 / 8h for 2-3 weeks to induce callus.

[0048] (5) Redifferentiation: The selected explants are transferred to differentiation medium M3 and subcultured every 2-3 weeks. During this period, the browned and dead explants are eliminated. The explants with swollen ends and green spots are preferred.

[0049] (6) Bud growth and rooting: After the differentiated buds have formed into plant forms, they are cut with a scalpel and placed in M4 culture medium for 4 weeks. After rooting, they can be hardened in a light incubator and then transferred to a greenhouse for cultivation. During the rooting process, if multiple plants of the same transformant are to be obtained, the growth time can be extended and the plants can be cut into multiple plant segments containing leaf buds for tissue culture.

[0050] 3. Identification of positive rapeseed seedlings The OE-CP15A transgenic rapeseed that had undergone resistance screening was used to extract DNA from the leaves of the transgenic rapeseed using the CTAB method. When amplified using the designed PCR specific primers BnaCP15A-F and M13-48R, specific DNA fragments were amplified. However, when using non-transformed rapeseed genomic DNA as a template, no fragments were amplified, indicating that BnaCP15A-positive rapeseed seedlings had been obtained.

[0051] Furthermore, the positive rapeseed seedlings identification results are as follows Figure 6 shown.

[0052] Example 5: Identification of salt tolerance of plants overexpressing the BnaCP15A gene at the seedling stage To investigate the function of the BnaCP15A gene under salt stress, transgenic and wild-type seeds (Westar) were germinated for one week and then transferred to 1 / 4 Hoagland nutrient solution. Two weeks later, the seedlings were treated with 200 mM NaCl solution. Phenotypic observations were performed after two weeks of stress, and the fresh and dry weights were calculated.

[0053] (1) Plant phenotypes such as Figure 7 As shown in the figure, under normal conditions, there was no significant difference between the transgenic plants and the wild type, but under salt stress, the growth potential of the transgenic plants was significantly better than that of the wild type.

[0054] (2) Count the fresh weight of the plants. The results are as follows: Figure 8 As shown in the figure, under normal conditions, there was no significant difference in fresh weight between transgenic plants and wild type; under salt stress, salt stress significantly inhibited the growth of plants, but the fresh weight of transgenic plants was significantly higher than that of wild type, indicating that transgenic plants were less inhibited and overexpression of the BnaCP15A gene could improve the salt tolerance of rapeseed.

[0055] (3) Count the dry weight of the plants. The results are as follows: Figure 9 As shown in the figure, under normal conditions, there was no significant difference in dry weight between transgenic plants and wild type, but under salt stress, the dry weight of transgenic plants was significantly higher than that of wild type, indicating that transgenic plants were less inhibited by stress and overexpression of the BnaCP15A gene could improve the salt tolerance of rapeseed.

[0056] The above results show that overexpression of the BnaCP15A gene significantly improves the salt tolerance of Brassica napus, effectively reduces the damage to salt-stressed plants, and reduces biomass loss. The BnaCP15A gene can positively regulate the salt tolerance of Brassica napus.

Claims

1. Application of a protein encoded by the BnaCP15A gene of Brassica napus in improving plant salt tolerance, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that The plant is rapeseed.

3. The application according to claim 1, characterized in that The improvement of plant salt tolerance is achieved by increasing the expression level of the BnaCP15A gene in the plant.

4. The application according to claim 3, characterized in that The method for increasing the expression level of the BnaCP15A gene in plants comprises: constructing a plant expression vector for expressing the BnaCP15A gene, and transforming the plants.

5. The application according to claim 4, characterized in that: The backbone vector of the plant expression vector is pCAMBIA2300.

6. The application according to claim 4, characterized in that: The transformation was carried out using Agrobacterium-mediated method.

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