A StUGT178 gene for enhancing potato salt stress resistance, its vector, recombinant bacteria, cells, and applications.
By enhancing the salt stress resistance of potatoes through the StUGT178 gene, the negative impact of salt stress on potato growth in existing technologies has been resolved, resulting in enhanced growth and development and improved quality of plants under salt stress conditions.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ANHUI AGRICULTURAL UNIVERSITY
- Filing Date
- 2024-07-19
- Publication Date
- 2026-05-26
AI Technical Summary
Existing technologies are insufficient to effectively improve the salt stress resistance of potatoes, thus affecting their yield and quality.
Using the StUGT178 gene, the plant's salt stress tolerance was enhanced by increasing proline content and antioxidant enzyme activity, and decreasing malondialdehyde content. Genetic engineering was carried out using recombinant vectors and recombinant bacteria.
It significantly improved the growth and development of plants under salt stress conditions and enhanced their tolerance to salt stress, providing a theoretical basis for breeding stress-resistant potato varieties.
Smart Images
Figure CN118910103B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant genetic engineering technology, and in particular to a StUGT178 gene that enhances the salt stress resistance of potatoes, its vector, recombinant bacteria, cells, and applications. Background Technology
[0002] Potato (Solanum tuberosum L.) belongs to the genus Solanum in the family Solanaceae and is one of the world's most important food crops.
[0003] Salt stress is one of the most common environmental stresses that negatively impacts plant growth and development, and can severely affect potato yield and quality. Therefore, identifying salt-tolerant functional genes in potatoes and gaining a deeper understanding of their mechanisms of action is of great significance for developing salt-resistant potato varieties through molecular breeding, thereby increasing potato yield. Summary of the Invention
[0004] The technical problem to be solved by the present invention is to provide a StUGT178 gene for enhancing potato salt stress resistance, its vector, recombinant bacteria, cells, and applications.
[0005] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:
[0006] A StUGT178 gene that enhances the salt stress resistance of potatoes, the nucleotide sequence of which is shown in SEQ ID NO.1.
[0007] As one of the preferred embodiments of the present invention, the cloning steps of the StUGT178 gene are as follows: extracting total RNA from potato and reverse transcribing it into cDNA; designing specific amplification primers StUGT178-F and StUGT178-R; performing PCR amplification using cDNA as a template to obtain the target gene StUGT178; wherein, the sequences of the specific amplification primers StUGT178-F and StUGT178-R are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.
[0008] As one of the preferred embodiments of the present invention, the StUGT178 gene enhances the plant's salt stress tolerance by increasing the proline content and antioxidant enzyme activity of the plant under salt stress conditions, reducing the malondialdehyde content.
[0009] A recombinant vector containing the StUGT178 gene described above.
[0010] As one of the preferred embodiments of the present invention, the recombinant vector is specifically pRI101-35S-StUGT178-GFP, and the construction method is as follows: the StUGT178 gene sequence is inserted into the Sal I and BamHI restriction sites of the vector pRI101-35S-GFP using homologous recombination.
[0011] A recombinant bacterium containing the aforementioned StUGT178 gene.
[0012] As one of the preferred embodiments of the present invention, the recombinant vector pRI101-35S-StUGT178-GFP constructed from the StUGT178 gene is introduced into bacterial cells to obtain the product.
[0013] A genetically engineered host cell, wherein the host cell genome integrates an exogenous StUGT178 gene with a sequence as shown in SEQ ID NO.1, or a plant overexpression vector constructed from the host cell containing the StUGT178 gene.
[0014] An application of the StUGT178 gene described above in the cultivation of transgenic plants resistant to salt stress.
[0015] The advantages of this invention compared to the prior art are as follows: the StUGT178 gene provided by this invention can increase the proline content and antioxidant enzyme activity of plants under salt stress conditions, and reduce the malondialdehyde content, thereby enhancing the salt stress tolerance of plants and providing a theoretical basis for breeding stress-resistant potato varieties. Attached Figure Description
[0016] Figure 1 The images show the semi-quantitative and quantitative PCR detection results of wild-type plants and transgenic plants in Example 4 (Figure A is a semi-quantitative PCR detection gel image, and Figure B is a quantitative PCR detection result image; where WT and CK are wild-type control plants, and OE1-11 are transgenic plants obtained through screening).
[0017] Figure 2 The germination rates of wild-type and overexpressing transgenic Arabidopsis thaliana under different NaCl concentrations in Example 4 are shown in Figure 4. (Figure A shows the germination of plants under different NaCl concentrations, and Figures B to E show the statistical germination rates of plants under different NaCl concentrations; where WT is the wild-type control and OE3 / 4 / 6 are transgenic plants.)
[0018] Figure 3Example 4 shows the taproot lengths of wild-type and overexpressing transgenic Arabidopsis thaliana under different NaCl concentrations (Figure A shows the taproot lengths of plants under different NaCl concentrations, and Figures B to E show the taproot lengths of plants under different NaCl concentrations; where WT is the wild-type control and OE3 / 4 / 6 are transgenic plants).
[0019] Figure 4 The phenotypes of wild-type and overexpressing transgenic Arabidopsis thaliana after salt treatment in Example 4 are shown in the figure (WT is the wild-type control, OE3 / 4 / 6 is the transgenic plant, 200mM represents 200mM NaCl salt treatment, and 0mM represents 0mM NaCl control).
[0020] Figure 5 This is a comparison of proline and malondialdehyde (MDA) content in wild-type and overexpressing transgenic Arabidopsis thaliana before and after salt stress treatment in Example 4 (Figure A shows the proline content results, and Figure B shows the MDA content results; where WT is the wild-type control, OE3 / 4 / 6 are transgenic plants, before stress represents normal growth conditions, after stress represents 200mM NaCl treatment for 5 days, and "**" indicates p<0.01).
[0021] Figure 6 The results of POD and CAT activity analysis of wild-type and overexpressing transgenic Arabidopsis thaliana before and after salt stress treatment in Example 4 are shown in Figure A (Plot A shows POD activity results, and Figure B shows CAT activity results; where WT is the wild-type control, OE3 / 4 / 6 are transgenic plants, before stress represents normal growth conditions, after stress represents 200mM NaCl treatment for 5 days, and "**" indicates p<0.01). Detailed Implementation
[0022] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the reagents and experimental methods used in the following embodiments are all conventional reagents or methods in the art and will not be described again.
[0023] Example 1, StUGT178 gene:
[0024] The nucleotide sequence of the StUGT178 gene is shown in SEQ ID NO.1.
[0025] Gene cloning steps:
[0026] (1) Potato "DM" material test-tube seedlings were grown in a light incubator. After 4 to 5 weeks of growth, leaf samples were taken and immediately placed in liquid nitrogen and stored at -80℃.
[0027] (2) Leaf RNA extraction and cDNA synthesis were performed using the corresponding kits.
[0028] (3) The StUGT178 gene sequence is shown in SEQ ID NO.1. Based on the target gene CDS sequence, specific amplification primers StUGT178-F and StUGT178-R were designed, and their sequences are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.
[0029] (4) Using cDNA as a template, PCR amplification was performed using a high-protection enzyme. The amplification system consisted of 1 μL of cDNA, 0.2 μL of GXL DNA Polymerase, 0.4 μL each of specific primers StUGT178-F and StUGT178-R, 1.6 μL of dNTP Mix, 4 μL of 5×Buffer, and 12.4 μL of ddH2O. The amplification program was as follows: 98℃ pre-denaturation for 2 min, 98℃ denaturation for 10 s, 58℃ annealing for 30 s, and 68℃ extension for 80 s, for a total of 38 cycles.
[0030] (5) Perform agarose gel electrophoresis on the PCR amplification products, and use a gel recovery kit to cut and recover the target fragment to obtain the target gene StUGT178 fragment.
[0031] (6) The target fragment was constructed into the pMD19 vector, transformed into E. coli, and the target gene sequence was obtained after sequencing by the company.
[0032] Example 2: Construction of the recombinant vector pRI101-35S-StUGT178-GFP:
[0033] (1) Design a pair of adapter primers based on the pRI101 vector sequence and restriction sites, as shown in SEQ ID NO.4 and SEQ ID NO.5; use high-protection enzyme to amplify the target fragment with adapter, and the amplification system and procedure are the same as those for gene cloning.
[0034] (2) The pRI101 vector was double-digested with restriction endonucleases Sal I and BamHI to obtain a linearized vector.
[0035] (3) The target fragment with adapter and the linearized vector fragment were recovered using a gel recovery kit, and the target fragment was ligated to the pRI101 vector using a homologous recombination kit (purchased from Hanheng Biotechnology Co., Ltd.); Ligation system: 0.8 μL of StUGT178 adapter primer amplification fragment, 1.2 μL of linearized pRI101 vector, 5 μL of HB-infusion Master Mix (2x), and ddH2O to 10 μL; Reaction program: 50℃ for 20 min, and stored at 4℃.
[0036] Example 3: Construction of recombinant bacteria:
[0037] The ligation product from Example 2 was transferred into Escherichia coli DH5α, specifically as follows:
[0038] (1) Take the competent state out of the -80℃ freezer and place it on ice to thaw.
[0039] (2) Take 10 μL of the ligation product into the competent state and gently aspirate and mix.
[0040] (3) Ice bath for 25 minutes, then heat shock at 42℃ for 45 seconds, then ice bath for 3 minutes.
[0041] (4) Add 250 μL of antibiotic-free LB medium to a clean bench, mix well, and then incubate at 37°C in a shaker for 1 h.
[0042] (5) Take 100 μL of reaction solution and spread it on a solid culture medium containing kanamycin. Invert the plate and incubate overnight at 37°C.
[0043] Example 4: Obtaining Arabidopsis thaliana lines overexpressing StUGT178 and salt stress test:
[0044] (1) The recombinant bacteria obtained in Example 3 were extracted using a plasmid extraction kit and transformed into Agrobacterium GV3101.
[0045] (2) Arabidopsis thaliana was infected by dipping flowers and T0 generation seeds were harvested.
[0046] (3) Transgenic plants of generation T1 and T2 were screened and identified by MS medium containing kanamycin, semi-quantitative and real-time PCR.
[0047] Figure 1 The results show semi-quantitative and real-time quantitative PCR detection of wild-type and transgenic plants (WT represents wild-type control plants, and OE1–11 represent transgenic plants selected from resistance plates). Figure 1It can be seen that the transcription level of the StUGT178 gene in the overexpressing transgenic Arabidopsis thaliana lines is significantly higher than that in the wild type (WT), indicating that the genetic transformation of transgenic Arabidopsis thaliana is successful and can be used for subsequent experiments.
[0048] (4) The obtained T3 generation homozygous transgenic lines (taking the screened OE3, OE4, and OE6 transgenic plants as examples) were used for salt stress treatment, phenotypic observation, and physiological index determination.
[0049] Observation results: After treatment with different concentrations of salt stress, the germination rate and taproot length of the transgenic plants (OE3 / 4 / 6) were significantly higher than those of the wild-type plants. Figure 2 and Figure 3 As shown; When Arabidopsis thaliana overexpression and wild-type plants grown in soil were treated with salt stress (200 mM NaCl), the leaves of the transgenic plants (OE3 / 4 / 6) were more spread out and the growth was relatively better than that of wild-type Arabidopsis thaliana. Figure 4 As shown; after salt stress treatment, the proline content in transgenic plants (OE3 / 4 / 6) was significantly higher than that in wild-type plants, while the malondialdehyde content was significantly lower than that in wild-type plants, such as... Figure 5 As shown; under salt stress conditions, transgenic plants (OE3 / 4 / 6) exhibited stronger antioxidant enzyme (POD and CAT) activity than wild-type plants, such as Figure 6 As shown.
[0050] In summary, the StUGT178 gene provided by this invention can increase the proline content and antioxidant enzyme activity of plants under salt stress conditions, and reduce the malondialdehyde content, thereby enhancing the salt stress tolerance of plants and providing a theoretical basis for breeding stress-resistant potato varieties.
[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method to enhance the salt stress resistance of potatoes StUGT178 The application of genes in the breeding of transgenic plants resistant to salt stress is characterized by, The StUGT178 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and it is obtained by overexpressing the gene. StUGT178 The gene enhances the proline content and antioxidant enzyme activity of plants under salt stress, reduces malondialdehyde content, and improves the salt stress tolerance of plants; the plant is Arabidopsis thaliana.