CaGGP-2 gene for regulating and controlling salt resistance of plants and application of CaGGP-2 gene
By introducing the CaGGP-2 gene into plants and constructing a recombinant expression vector, the salt tolerance of peppers was improved, solving the problem of limited growth of peppers under salt stress and achieving significant enhancement of salt tolerance and antioxidant enzyme system.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-26
- Publication Date
- 2026-03-31
AI Technical Summary
Chili peppers are restricted in their growth under salt stress. Ion imbalance and osmotic pressure affect nutrient absorption and metabolism, and existing technologies are insufficient to effectively improve their salt tolerance.
By introducing the CaGGP-2 gene from chili peppers, a recombinant expression vector was constructed and transformed into plants to improve their salt tolerance.
It significantly improved the salt tolerance of plants, reduced chlorophyll degradation and membrane lipid peroxidation caused by salt stress, enhanced the responsiveness of antioxidant enzyme systems, and improved the growth adaptability of plants under salt stress.
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Figure CN121759477A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant genetic engineering technology, and in particular to a CaGGP-2 gene that regulates plant salt tolerance and its applications. Background Technology
[0002] Chili peppers, as an important economic crop, are widely cultivated around the world. They are rich in vitamins, various essential minerals, phytochemicals, and dietary fiber. They not only add flavor to food, but more importantly, they have health potential in promoting antioxidation, anti-inflammation, metabolic regulation, and cardiovascular protection.
[0003] However, salt stress is one of the common and serious abiotic stresses in the growth of peppers. Under salt stress, high concentrations of sodium and chloride ions in the soil can cause ion imbalance and osmotic pressure in peppers, thereby affecting nutrient absorption and metabolism, leading to restricted growth and development.
[0004] Therefore, studying gene mining technology to address the impact of salt stress on chili pepper growth is crucial for improving the adaptability and economic benefits of chili pepper cultivation. Summary of the Invention
[0005] To address the problems existing in the prior art, this invention provides a CaGGP-2 gene for regulating plant salt tolerance and its application. Overexpression of the CaGGP-2 gene in plants can significantly improve plant salt tolerance. The specific technical solution is as follows: The present invention provides a CaGGP-2 gene, which is derived from pepper (Capsicumannuum), and the nucleotide sequence of the gene is (1), (2) or (3). (1) The nucleotide sequence shown in SEQ ID NO:3; (2) The nucleotide sequence encoded by hybridization with the nucleotide sequence shown in SEQ ID NO:3 under stringent conditions; (3) A nucleotide sequence that has more than 70% homology with the nucleotide sequence defined in (1) or (2) and encodes a nucleotide sequence; preferably having 75% homology, more preferably having 80% homology, more preferably having 85% homology, more preferably having 90% homology, more preferably having 95% homology, more preferably having 96% homology, more preferably having 97% homology, more preferably having 98% homology, and most preferably having 99% homology.
[0006] The present invention also provides a protein encoded by the CaGGP-2 gene as described above, wherein the amino acid sequence of the protein is (a) or (b). (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:4; (b) A protein derived from SEQ ID NO:4 with the amino acid residue sequence described in SEQ ID NO:4 having one or more amino acid residues substituted and / or deleted and / or added, and having the same function.
[0007] The sequence SEQ ID NO:3 in the sequence listing consists of 1323 bases and encodes the protein whose amino acid sequence is shown in the sequence SEQ ID NO:4 in the sequence listing.
[0008] Expression cassettes, recombinant expression vectors, transgenic cell lines, or recombinant bacteria containing the coding gene of the protein are also within the scope of protection of this invention.
[0009] Furthermore, the recombinant expression vector is obtained by inserting the CaGGP-2 gene into the multiple cloning site of the initial vector pCAMBIA1300-35S-GFP.
[0010] Furthermore, the recombinant expression vector is obtained through the following steps: the initial vector pCAMBIA1300-35S-GFP is obtained through the following steps: Step 1: The initial vector pCAMBIA1300-35S-GFP was processed... Bam HI and Xba I. Double enzyme digestion to recover the large fragment of the vector; Step 2: Ligate the large vector fragment recovered in Step 1 with the target gene fragment to obtain the recombinant expression vector.
[0011] Primer pairs for amplifying the full length of the chili CaGGP-2 gene or any fragment thereof described above are also within the scope of protection of this invention. The primer pair includes an upstream primer F and a downstream primer R. The nucleotide sequence of the upstream primer F is shown in SEQ ID NO:1, and the nucleotide sequence of the downstream primer R is shown in SEQ ID NO:2.
[0012] The application of the above-mentioned chili CaGGP-2 gene or protein or recombinant expression vector, expression cassette, transgenic cell line or recombinant bacteria in improving plant salt tolerance also falls within the scope of protection of this invention.
[0013] Furthermore, the plant is a dicotyledonous plant.
[0014] Preferably, the plant is chili pepper or Arabidopsis thaliana.
[0015] The present invention also provides a method for cultivating transgenic plants, wherein the CaGGP-2 gene described above is introduced into a recipient plant to obtain a transgenic plant; compared with wild-type plants, the salt tolerance of the transgenic plant is improved; the plant is a dicotyledonous plant.
[0016] Preferably, the plant is chili pepper or Arabidopsis thaliana.
[0017] Preferably, the CaGGP-2 gene described above is introduced into the leaves of the recipient plant.
[0018] Furthermore, the CaGGP-2 gene is introduced into the recipient plant via the recombinant expression vector described above.
[0019] Beneficial Effects: Compared with existing technologies, the specific beneficial effects of this invention are as follows: This invention provides a CaGGP-2 gene, and the application of CaGGP-2 or its transgenic biological materials can improve the salt tolerance of plants. Experimental results show that the transgenic plants obtained by introducing the CaGGP-2 gene into recipient plants exhibit significantly higher salt tolerance than wild-type plants. Therefore, the CaGGP-2 protein and its encoding gene provided by this invention have significant application value in improving plant salt tolerance and in cultivating salt-tolerant plant varieties. It provides important evidence for research on improving plant salt tolerance, is of great significance for increasing crop yields, and has broad application space and market prospects in the agricultural field. Attached image description:
[0020] Figure 1 Agarose gel electrophoresis image of the CaGGP-2 gene; Figure 2 The tertiary structure of the protein encoded by the aGGP-2 gene for ChimeraX was constructed. Figure 3 The expression of CaGGP-2 in chili peppers under salt stress; Figure 4 Agarose gel electrophoresis image of CaGGP-2 transgenic plants; Figure 5 The results show the salt tolerance of CaGGP-2 transgenic plants and wild-type plants. Detailed Implementation
[0021] To facilitate understanding of the present invention, a more comprehensive description will be given below with reference to embodiments, and preferred embodiments of the present invention are provided below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. The embodiments are for illustrative purposes only and are not intended to limit the present invention. Modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the present invention are all within the scope of the present invention. The purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.
[0022] Example 1: Cloning of the CaGGP-2 gene in chili pepper 1. Seeds of the chili variety 'Xianhong No. 1' were germinated at 25°C, and seedlings were cultivated in a light-incubator using the floating seedling method after 2 days. The culture solution was the field trial nutrient solution with pH 6.0, day and night temperature of 25°C / 18°C, relative humidity of 60%~70%, and light intensity of 6000 lx.
[0023] 2. RNA isolation: RNA extraction was performed according to TIANGEN. ® Extract according to the kit instructions.
[0024] 3. Reverse transcription to synthesize cDNA libraries cDNA library synthesis is based on abm ® The All-in-One 5X RT MasterMix was synthesized according to the instructions, and the reaction system is as follows:
[0025] Incubate the mixture at 37°C for 15 minutes, then continue incubating at 60°C for 10 minutes. The prepared cDNA can be used immediately or stored at -20°C.
[0026] 4. Amplification of the coding region (CDS) of the CaGGP-2 gene in chili peppers The coding sequence (CDS) of chili pepper CaGGP-2 (gene number: Capana02g002879) was obtained by searching chili pepper genome and full-length gene databases. PCR amplification primers were designed based on the predicted information. The primer sequences are as follows: CaGGP-2-F: 5'-gagaacacgggggactctagaATGTTGACTATAAAGAGGGTGCCTACA-3'(SEQID NO:1) CaGGP-2-R: 5'-gcccttgctcaccatggatccTCAGTGTAGCACCAAACAATCTTGAGG-3'(SEQID NO:2) The CaGGP-2 gene cloning reaction system is as follows:
[0027] The CaGGP-2 gene CDS was obtained by direct cloning from cDNA. Agarose gel electrophoresis was used to detect the PCR amplification products, yielding an amplified fragment of 1323 bp. Figure 1 Based on the results of the above steps, the target cDNA sequence was obtained, and its nucleotide sequence is shown in SEQ ID NO:3 in the sequence listing. This gene was named CaGGP-2.
[0028] Example 2: Protein sequence information and physicochemical property analysis of the protein encoded by the chili pepper CaGGP-2 gene. The amino acid sequence of the protein encoded by the chili pepper CaGGP-2 gene (gene number: Capana02g002879) was deduced using DNAMAN software. The protein consists of 440 amino acids and has a molecular weight of 49.70 kDa. The tertiary structure of the protein was constructed using ChimeraX software. Figure 2 The physicochemical properties of the protein were analyzed. The isoelectric point was 5.79, indicating that the protein was acidic. The instability index was 47.78, and the aliphatic index was 92.09. Detailed information is shown in the sequence SEQ ID NO:4 in the sequence listing.
[0029] Example 3: Expression analysis of CaGGP-2 gene in chili peppers under salt treatment When the chili pepper plants have 5 leaves and 1 bud, select healthy plants of uniform size for treatment, and add NaCl to the nutrient solution to achieve a NaCl concentration of 200 mmol·L⁻¹. -1 The qRT-PCR primers for the CaGGP-2 gene were (SEQ ID NO:5, SEQ ID NO:6). Results showed that CaGGP-2 expression was upregulated by approximately 1.5-fold after 12 h of salt treatment in this invention. Figure 3 The primer sequences are: CaGGP-2-F: 5'-GGAAGATACTGGCAAGGA-3' (SEQ ID NO: 5) CaGGP-2-R: 5'-CACTGAGCAATGAGAATGTT-3' (SEQ ID NO: 6) Example 4: Construction of Transgenic Plants 1. Vector linearization use Bam HI and Xba The vector was linearized by double digestion with restriction endonucleases. The system was as follows:
[0030] 2. Target fragment recombination and E. coli transformation The connection between the target fragment and the carrier was referenced from CloneUFO of Nanjing Jujiang Company. ® Operation of the recombinant cloning kit. The reaction system is as follows:
[0031] After the system is prepared, gently pipette to mix all components. Incubate at 37°C for 30 min. After the reaction is complete, immediately place the reaction tube in an ice-water bath to cool for 5 min. Then store at -20°C and thaw for conversion when needed.
[0032] The transformation of E. coli was performed according to the instructions for DH5α chemocompetent cells from Nanjing Jujiang Company, and the specific procedures are as follows: Remove the DH5α competent cells from -80 ℃ and quickly insert them into ice. After 15 min, wait for the bacterial block to melt, then add 10 μL of the ligation product while rotating the pipette tip (5 turns clockwise) to gently mix. Avoid using the pipette to agitate the cells. Let the mixture stand in ice for 30 min.
[0033] Heat shock at 42℃ for 90 seconds, then quickly return to ice and let stand for 5 minutes. Shaking will reduce the conversion efficiency.
[0034] Add 800 μL of antibiotic-free sterile LB medium to a centrifuge tube, mix well, and incubate at 37 °C and 200 rpm for 45 min.
[0035] Centrifuge at 3500 rpm for 3 min to collect the bacteria. Aspirate 800 μL, leaving about 100 μL of supernatant. Gently pipette the bacterial block to resuspend it and spread it onto LB medium containing kanamycin.
[0036] Invert the plate and incubate it overnight at 37°C for 12 hours.
[0037] 3. Expansion culture of positive clone strains and plasmid extraction Select round, smooth, and uniform bacterial strains from overnight culture plates and place them into centrifuge tubes containing 20 μL of liquid LB medium (containing kanamycin). Using the bacterial culture as a template, PCR verification was performed using CaGGP-2 primers (SEQ ID NO:1, SEQ ID NO:2) to screen for positive strains containing the target gene. The positive strains were then placed in LB medium containing kanamycin and cultured in a shaker at 37°C and 200 rpm for 12 h.
[0038] The turbid bacterial solution was prepared according to TIANGEN ® Instructions for use of the high-purity plasmid extraction kit.
[0039] 4. Agrobacterium transformation and identification of positive strains Remove GV3101 Agrobacterium competent cells and insert them into ice for 5-10 min (store in an ultra-low temperature freezer at -80℃) until they thaw.
[0040] Add 6 μL of the target gene plasmid to a centrifuge tube, mix gently, and incubate on ice for 10 min.
[0041] Quick-freeze in liquid nitrogen for 5 min, then in a water bath at 37°C for 5 min, followed by an ice bath for 5 min.
[0042] Add 700 μL of antibiotic-free LB liquid culture medium to each tube in the clean bench and incubate at 28°C and 220 rpm for 2-3 h.
[0043] Centrifuge at 6000 rpm for 1 min to collect the bacteria. Discard 700 μL of supernatant in a clean bench, and keep about 100 μL. Gently pipette the resuspended bacterial solution and spread it on LB solid medium containing Kana and Rif. After drying, invert the medium and incubate at 28℃ for 2-3 days.
[0044] Select single colonies with round and smooth shapes and suspend them in 20 μL LB medium for PCR verification using CaGGP-2 primers (SEQ ID NO:1, SEQ ID NO:2).
[0045] Add the correct bacterial culture to 50 mL of LB medium containing Kana and Rif, and incubate at 28°C with shaking at 200 rpm for 24 h.
[0046] 5. Arabidopsis thaliana infection and screening The specific steps of Arabidopsis thaliana infection are as follows: Agrobacterium-mediated culturing to OD 600 The absorbance is 1.0-1.2.
[0047] Centrifuge at 5000 g for 10 min, remove the supernatant, and retain the bacterial cells.
[0048] The bacterial cells were resuspended in a transformation medium with the same volume as the centrifuged bacterial culture.
[0049] Remove the siliques and flowers from the Arabidopsis thaliana plants to be transformed.
[0050] Sillwet-77 Arabidopsis flowers were added to the resuspended bacterial solution and completely immersed in the solution. The vacuum was then applied to 380 mmHg for 5 min, followed by incubation in a 22°C incubator for 24 h in the dark. The final concentration was 0.025%.
[0051] The Arabidopsis thaliana was then placed in a normal light incubator for growth, and the seeds were harvested for selection.
[0052] After the collected transgenic Arabidopsis seeds (designated as generation T0) were dried, they were evenly sown on resistant 1 / 2 MS medium (containing 40 μL hygromycin, 80 μL termethin, and 80 μL carbenicillin) in a clean bench. Wash the seeds with 75% alcohol for 1 minute, shaking them occasionally to remove as much alcohol as possible.
[0053] Clean the alcohol off with sterile water, then dry it as much as possible.
[0054] Wash the seeds with 10% NaClO solution for 5 minutes, shaking repeatedly.
[0055] Rinse five times with sterile water, absorbing as much water as possible each time.
[0056] Evenly transfer water and Arabidopsis seeds into 1 / 2 MS medium containing resistance, remove excess water, then dry in a clean bench with air, seal and vernalize at -4℃ for 2-3 days, then transfer to an incubator for culture.
[0057] The emerging Arabidopsis seedlings were designated as Generation T1, and seeds from individual plants were collected and designated as Generation T1 seeds. The harvested Generation T1 seeds were further screened using 1 / 2 MS resistance medium to obtain positive Generation T2 plants. The obtained Generation T2 transgenic seedlings were then processed using TIANGEN... ® RNA was extracted using a plant RNA extraction kit, and positive plants were verified by PCR using CaGGP-2 primers (SEQ ID NO:1, SEQ ID NO:2). PCR amplification products were detected by agarose gel electrophoresis. Figure 4 ).
[0058] Example 5: Salt Tolerance Analysis of Transgenic Plants Wild-type Arabidopsis seeds and transgenic Arabidopsis seeds infused with the CaGGP-2 gene were cultured separately on 1 / 2 MS medium. When seedlings reached 4 weeks of age, they were subjected to gradient salt stress treatment: first, the plants were immersed in a 50 mM salt solution for 2 days, then transferred to a 100 mM salt solution for another 2 days, and finally placed in a 200 mM salt solution. Plant phenotypes were observed three weeks after the salt treatment, and relative chlorophyll content, relative conductivity, malondialdehyde (MDA) content, and the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were measured.
[0059] Experimental results showed that, under normal growth conditions, the phenotypes of wild-type plants and CaGGP-2 transgenic plants were basically identical. Figure 5 A). However, after salt stress treatment, the yellowing of leaves in wild-type plants was significantly more severe than in transgenic plants (A). Figure 5 A). Physiological indicators showed that under salt stress, the relative chlorophyll content of both types of plants decreased compared to the control, but the content of transgenic plants was higher than that of wild-type plants. Figure 5 B); Relative conductivity increased after salt stress, with the increase being greater in wild-type plants than in transgenic plants. Figure 5 C); MDA content also increases due to salt stress, and the accumulation is higher in wild-type plants ( Figure 5 D). Regarding antioxidant enzyme activity, salt stress increased SOD activity, and the SOD activity of transgenic plants was higher than that of wild-type plants. Figure 5E); while the activities of CAT and POD both decreased after salt stress, but the transgenic plants still maintained higher activity levels than the wild type ( Figure 5 F, Figure 5 G).
[0060] The above results indicate that the introduction of the CaGGP-2 gene can effectively alleviate chlorophyll degradation, membrane lipid peroxidation, and membrane damage caused by salt stress, and enhance the responsiveness of the antioxidant enzyme system, thereby positively regulating the salt tolerance of Arabidopsis thaliana. Therefore, the CaGGP-2 gene has clear application potential in improving crop salt tolerance.
[0061] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.
Claims
1. The CaGGP-2 gene, characterized by, The gene is derived from chili pepper, and the nucleotide sequence of the gene is (1), (2) or (3). (1) The nucleotide sequence shown in SEQ ID NO:3; (2) The nucleotide sequence encoded by hybridization with the nucleotide sequence shown in SEQ ID NO:3 under stringent conditions; (3) A nucleotide sequence that has more than 70% homology with the nucleotide sequence defined in (1) or (2) and encodes a nucleotide sequence.
2. The protein encoded by the CaGGP-2 gene as described in claim 1, characterized in that, The amino acid sequence of the protein is (a) or (b); (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:4; (b) A protein derived from SEQ ID NO:4 with the amino acid residue sequence described in SEQ ID NO:4 having one or more amino acid residues substituted and / or deleted and / or added, and having the same function.
3. A recombinant expression vector, expression cassette, transgenic cell line, or recombinant bacteria containing the CaGGP-2 gene as described in claim 1.
4. The recombinant expression vector according to claim 3, characterized in that: The recombinant expression vector was obtained by inserting the CaGGP-2 gene into the multiple cloning site of the initial vector pCAMBIA1300-35S-GFP.
5. A primer pair for amplifying the full-length CaGGP-2 gene or any fragment thereof as described in claim 1, characterized in that, The primer pair includes an upstream primer F and a downstream primer R, the nucleotide sequence of which is shown in SEQ ID NO:1 and the nucleotide sequence of which is shown in SEQ ID NO:
2.
6. The application of the CaGGP-2 gene as described in claim 1, the protein as described in claim 2, or the recombinant expression vector, expression cassette, transgenic cell line, or recombinant bacteria as described in claim 3 in improving plant salt tolerance, characterized in that... The plant in question is a dicotyledonous plant.
7. A method for cultivating transgenic plants, characterized in that, The CaGGP-2 gene of claim 1 is introduced into a recipient plant to obtain a transgenic plant; compared with the wild-type plant, the salt tolerance of the transgenic plant is improved; the plant is a dicotyledonous plant.
8. The method according to claim 7, characterized in that: The CaGGP-2 gene is introduced into the recipient plant via the recombinant expression vector as described in claim 3 or 4.