ChtSGAT gene of dendrobenthamia tonkinensis and application of ChtSGAT gene in improvement of plant salt tolerance

Abstract

The invention discloses application of a dendrobenthamia dogwood ChtSGAT gene in improvement of plant salt tolerance, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the ChtSGAT gene is as shown in SEQ ID NO. 1, and the amino acid sequence of the encoded protein of the ChtSGAT gene is as shown in SEQ ID NO. 2. By analyzing phenotypic growth, stress physiology and transcriptome response of kousa dogwood under salt stress, salt-tolerant candidate genes mainly participating in photosynthetic pathways are screened out, a photorespiration key gene ChtSGAT is subjected to arabidopsis thaliana transgenic function verification, and the result shows that the ChtSGAT gene is remarkably related to the salt tolerance of transgenic arabidopsis thaliana. And the salt tolerance of the transgenic arabidopsis thaliana is improved. The invention provides a theoretical basis and related genes for salt stress resistant plant breeding.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to Tokyo tetrapoda SGAT Genes and their application in improving salt tolerance in plants. Background Art

[0002] The global soil salinization area has exceeded 1 billion hectares, of which about 20% of irrigated farmland has significantly reduced productivity due to secondary salinization. Salt stress inhibits plant growth through ion toxicity and osmotic stress, resulting in crop yield reduction. The area of ​​saline-alkali land in coastal and inland my country has reached 100 million hectares, which seriously threatens food security and ecological security.

[0003] Tokyo four-leaf holly Cornus hongkongensis subsp. tonkinensis ) is a plant of the Cornaceae family, mainly distributed in China, Japan, Vietnam, etc. It shows strong salt tolerance and leaf color diversity in coastal saline-alkali land. SGAT The molecular function of the gene in salt tolerance in woody plants is unknown, and there is a lack of genetic transformation and functional verification of the gene. Summary of the invention

[0004] In view of the above problems, the present invention cloned the SGAT The gene was confirmed by overexpression vector construction and transgenic Arabidopsis functional verification, which confirmed that the gene can improve plant salt tolerance through the photorespiration-proline metabolism synergistic pathway. The present invention provides gene resources for the development of saline-alkali land-adapted plants with both stress resistance and ornamental value.

[0005] The invention provides a kind of Tokyo four-leaf hollyhock SGAT Application of genes in improving salt tolerance in plants.

[0006] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions: In a first aspect, the present invention provides a ChtSGAT gene of Tokyo tetrapoda, the nucleotide sequence of which is shown in SEQ ID NO.1, specifically:

[0007] Furthermore, the present invention provides a protein encoded by the ChtSGAT gene, whose amino acid sequence is shown in SEQ ID NO.2, specifically: MDYVNGPGRNHLFVPGPVNIPEQVIRAMNRNNEDYRSPAIPAMTRTLLEDVKKIFKTTSGTPFLIPTTGTGAWESALTNTLSPGDRIVSFLIGQFSLLWIDQQQRLNFNVDVVESDWGQGANLEVLASKLAADTAHTIKAICIVHNETATGVTNNLSAVRKILDDYRHPALFLVDGVSSICALDFRMDEWGIDVALTGSQK ALLSLPTGMGIVCASPKALEASQTAKSVRVFFDWKDYLKFYKMGTFWPYTPSIQLLYGLRTALDLIFEEGLDNVIARHSRLGKATRLAVEAWGLKNCTQKEEWFSNTVTAVVVPPYIDSSEIVRRGWQRYNLSLGLGLNKVAGKVFRIGHLGNLNDLQLLGCLAGVEMILKDVGYPVKLGSGVAAACAYLQNTIPMIPSRI.

[0008] In a second aspect, the present invention provides a primer pair for amplifying the ChtSGAT gene, wherein the forward primer sequence SEQ ID NO.3 of the primer pair is ChtSGAT-F: 5'-AAAGGTGTTCAGGATAGGGC-3', and the reverse primer sequence SEQ ID NO.4 is ChtSGAT-R: 5'-AATGGTGTTCTGTAGGTACGC-3'.

[0009] In a third aspect, the present invention provides a recombinant vector containing the ChtSGAT gene.

[0010] In a fourth aspect, the present invention provides an application of a ChtSGAT gene in regulating the salt tolerance of a target plant.

[0011] Furthermore, the above application is specifically manifested in the enhancement effect of overexpression of the ChtSGAT gene of Tokyo odonata on the salt tolerance of the target plant.

[0012] Furthermore, the ChtSGAT gene of the Tokyo hollyhock was introduced into the target plant, and the salt tolerance of the obtained transgenic plant was better than that of the target plant.

[0013] Furthermore, the plant is Arabidopsis thaliana.

[0014] The beneficial effects of the present invention are: The present invention cloned the first SGAT Gene, through the construction of overexpression vector and transgenic Arabidopsis functional verification, the salt tolerance of transgenic Arabidopsis was significantly improved. Based on the above findings, the SGAT The genes can be used for genetic improvement of salt tolerance in agricultural and forestry plants or for breeding plant varieties with greater salt tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 For plants SGAT Multiple sequence alignment; Figure 2 Phylogenetic tree construction for ChtSGAT protein sequences; Figure 3 The results of the hydrophilicity analysis of ChtSGAT protein; Figure 4 The predicted result of conserved domain of ChtSGAT protein; Figure 5 for SGAT Effects of overexpression on germination and root length of transgenic Arabidopsis. (ac) are the germination index, germination rate and phenotype of Arabidopsis cultured in MS medium containing 0%, 0.3%, 0.5% and 0.7% salt for 8 days; (de) is the root length analysis of seedlings transferred from germination day 3 to salt-containing medium for 8 days. Data are mean ± standard deviation (n=5); the same letters in the bar graph indicate no significant difference between groups (P<0.05); Figure 6 Salt stress SGAT Effects of transgenic Arabidopsis phenotype and chlorophyll fluorescence parameters. (a) Plant phenotype; (b) Chlorophyll fluorescence parameters (Fv / Fm, Fv / F0). Data are mean ± SD (n = 3 biological replicates, 10 seedlings per replicate); error bars represent mean ± SD; *, **, *** indicate significant differences between WT and transgenic lines under the same treatment conditions (P < 0.05, 0.01, 0.001), respectively. Same as the figure below; Figure 7 Salt stress SGAT Effects of transgenic Arabidopsis on physiological responses. (a) MDA content; (b) H 2 O 2 Accumulation amount; (ce) antioxidant enzyme activity (SOD, CAT, POD); (fh) osmotic regulating substance (SS, SP, Pro) content. DETAILED DESCRIPTION

[0016] In order to facilitate a better understanding of the present invention, the invention will be further described below in conjunction with some embodiments of the present invention, but the protection scope of the present invention is not limited to the following embodiments. Without departing from the spirit and essence of the present invention, modifications or replacements made to the methods, steps or conditions of the present invention all belong to the scope of the present invention. The test methods in the following embodiments that do not specify specific conditions are all conventional methods.

[0017] Plant materials and growth conditions The test plants were one-year-old seedlings of Tokyo odonata. Seedlings with similar growth and good growth conditions were selected and planted in a 50 L hydroponic box (length × width × height: 110 cm × 57 cm × 16 cm). 50 L of 1 / 2 Hoagland modified nutrient solution was added to the hydroponic box, aerated for 24 hours, set the pH to 6.0, replaced the nutrient solution every 7 days, and configured an aeration system to ensure dissolved oxygen.

[0018] Seeds of Arabidopsis ecotype Columbia were surface sterilized with 10% sodium hypochlorite for 20 min and then washed three times with sterile water. The sterilized seeds were inoculated on 1 / 2 MS medium and stored in a refrigerator at 4°C for 3 days before germination. The 7-day-old seedlings were transferred to a mixture of nutrient-rich soil and perlite (2:1) and grown in a growth chamber at 23°C with a photoperiod of 200 μmol m −2 s −1 , the photoperiod was 16 / 8 h (light / dark).

[0019] Embodiment 1: SGAT Gene cloning and analysis 1. SGAT Gene cloning Total RNA was extracted from young leaves of P. takyi using RNA prep Pure Plant Kit (Tiangen, China). The concentration and quality of RNA were detected by NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and 1% agarose gel electrophoresis, respectively. Total RNA was reverse transcribed using PrimeScript™II First Strand cDNA Synthesis Kit (Takara, Japan) to synthesize first-strand cDNA. Full-length primers were designed based on the transcriptome assembly sequence. The full-length primer sequences were: ChtSGAT-F: 5'-AAAGGTGTTCAGGATAGGGC-3' (SEQ ID NO.3) ChtSGAT-R: 5'-AATGGTGTTCTGTAGGTACGC-3' (SEQ ID NO.4) (1) Full-length sequence amplification Use a high-fidelity enzyme amplification system to obtain the full-length fragment. The PCR system is as follows: 5 PrimeSTAR Buffer (Mg 2+ Plus) 10 μL dNTP Mixture (2.5 mM each) 4 μL Primer F 1 μL Primer R 1 μL cDNA <200 ng PrimeSTAR HS DNA Polymerase 0.5 μL dH 2 O up to 50 μL The PCR reaction program was as follows: pre-denaturation at 94°C for 4 min; 35 cycles including: denaturation at 94°C for 30 s, annealing at 58°C for 40 s, extension at 72°C for 2 min; extension at 72°C for 10 min; and storage at 4°C.

[0020] (2) PCR product gel recovery The PCR product was recovered by 1% agarose gel electrophoresis using a gel recovery kit (Axygen, USA). The specific method is referred to the kit instructions. The vector connection is referred to the pClone007 SimpleVector Kit of Qingke Biotechnology.

[0021] (3) Competent state transformation Thaw the competent E. coli (Trans Chemically Competent Cell, Beijing Jinshi Column), add 10 μL of ligation solution to 100 μL competent cell. Ice bath for 30 min→heat shock at 42°C for 90 s→ice bath for 2 min→add 800 μL of LB medium without antibiotics, and recover at 37°C 220 rpm for 0.5-1 h. Centrifuge at room temperature for 3 min at 4000 rpm, keep 100 μL of bacterial solution for resuspending, apply Amp+ (100 μg / mL) plate, and invert and culture at 37°C for 12-16 h.

[0022] (4) Screening and detection of monoclonal bacteria Use a pipette to pick up a single white colony and place it in 10 μL sterile water. Mix by blowing and then take 1 μL and add it to 20 μL Super PCR Mix (green) and stir it slightly. Amplify according to the procedure of the pClone007 Simple Vector Kit, and send the positive bacterial PCR product to Nanjing Qingke Biotechnology for sequencing.

[0023] 2. Obtaining the complete ORF The obtained sequences were assembled using CAP3 Sequence Assembly Program (http: / / doua.prabi.fr / software / cap3) to obtain the complete sequence.

[0024] 3. SGAT Gene sequence analysis SGAT The full length of the gene ORF is 1206 bp, and the encoded protein consists of 401 amino acids, with a molecular weight of 44.09 kDa and an isoelectric point of 8.13.

[0025] Will SGAT The nucleotide sequences of the genes were compared with those of the genes in the NCBI-Blast database to explore the relationship between the genes and other species. In the NCBI database, highly homologous and representative species were selected for amino acid sequence homology comparison analysis. The analysis results were analyzed using Genedoc software, and the molecular phylogenetic tree between different species was constructed using mega software. The results are shown in the figure. Figure 1 and Figure 2 As shown, the results show SGAT The SGATs of other species are closely related and are speculated to have originated from the same ancestor. SGAT With blue fruit trees ( Nyssa sinensis ) (KAA8549846.1) and tea ( Camellia sinensis ) ( XP_028060239.1 ) sequences have the highest homology, with a similarity of up to 94.76%.

[0026] Select ProtScale analysis software in the ExPASy online website, paste the sequence on the ProtScale homepage, and select analysis to obtain the hydrophilicity and hydrophobicity analysis results of the protein. Overall, if the hydrophobic region is more than the hydrophilic region, it means that the hydrophobicity of the protein is greater than the hydrophilicity, and it may be a hydrophobic protein ( Figure 3 ).

[0027] By conducting conservative structure and prediction analysis on SGAT, we obtained the following results: Figure 4 shown. SGATThe protein encoded by the gene has the conservative structure of serine-glyoxylate aminotransferase and is the only member of superfamily cl30752. It contains three conserved sites of terpenoid synthase.

[0028] Embodiment 2: SGAT Functional identification of genes 1. Construction of transgenic plants Will SGAT The gene sequence was cloned into the pCAMBIA 2300-eGFP vector driven by the 35S:eGFP promoter. The wild-type Arabidopsis thaliana (Col-0) was transformed with the Agrobacterium tumefaciens GV3101 strain carrying the recombinant plasmid by the modified inflorescence dipping method. Positive strains were selected on 1 / 2 MS medium containing 50 mg / L kanamycin and screened by qRT-PCR after PCR verification. SGAT The third-generation homozygous transgenic lines with gene overexpression were used for subsequent experiments.

[0029] 2. Analysis of salt tolerance phenotype Three transgenic lines ( SGAT -OE1, SGAT -OE2, SGAT -OE3) and wild-type (WT) Arabidopsis seeds were placed on 1 / 2 MS medium containing 0%, 0.3%, 0.5%, and 0.7% salt concentrations, respectively, and after vernalization at 4°C for 3 days, they were germinated in a 22°C light incubator. Seed germination parameters were recorded within 24 hours, and the germination rate (GR) and germination index (GI) were calculated for 8 consecutive days: GR = (number of germinated seeds in each treatment / total number of seeds) × 100% GI = Σ (number of seeds germinated on day t / corresponding number of germination days) The 3-day-old seedlings were transferred to fresh medium containing the same salt concentration, and the root length was measured after 8 days. The WT and transgenic plants grown for 4 weeks were watered with 0.5% salt solution for 3 consecutive days. After 7 days, the Fv / Fm and Fv / F0 were measured using Handy PEA, and the leaves were collected to measure H 2 O 2 , MDA content and SOD, CAT, POD activity, and detect the content of osmotic regulating substances (SS, SP, Pro). 2 O 2 The accumulation was visualized by 3,3'-diaminobenzidine (DAB, CAS: 7411-49-6) staining (Bapatla et al., 2021), and the peroxidase (POD) activity was determined according to the method of Yin et al. (2018).

[0030] 3. SGAT Gene overexpression enhances salt tolerance in transgenic Arabidopsis By analyzing the germination characteristics of ChtSGAT overexpression lines (OE1, OE2, OE3) and wild type (WT) under salt stress, it was found that as the salt concentration increased, the germination of both WT and transgenic lines was inhibited ( Figure 5 a), but the germination index of the transgenic lines was always significantly higher than that of the WT ( Figure 5 b). Under salt-free conditions, the germination rate of the transgenic lines was superior to that of the WT only on the second day; under 0.3% and 0.5% salt concentrations, the transgenic lines showed significant advantages in the early germination period (1-2 days) (P<0.05) ( Figure 5 c), indicating that ChtSGAT overexpression may accelerate the early germination process of seeds. When the salt concentration reached 0.7%, the germination rate of the transgenic line was significantly higher than that of the WT from the third day of germination ( Figure 5 c). However, there was no significant difference in root length between WT and transgenic lines under different salt concentrations ( Figure 5 de).

[0031] Observation of 4-week-old WT and transgenic lines (OE) treated with 0.5% salt solution showed that: as the number of days of salt treatment increased, the growth of seedlings of both WT and OE lines was inhibited, but the overall growth state of the OE line was better than that of WT ( Figure 6 a). Under control conditions (CK), there was no significant difference in Fv / Fm (maximum photochemical efficiency) and Fv / F0 (potential activity of photosystem II) between OE and WT; however, under 0.5% salt stress, the Fv / Fm and Fv / F0 values ​​of the OE strain were significantly higher than those of the WT (P<0.05), indicating that the stability of its photosynthetic system was significantly enhanced ( Figure 6 b).

[0032] Under salt-free conditions, no obvious DAB staining signals were detected in the seedlings of WT and transgenic lines (H 2 O 2 However, under 0.5% salt stress, all plants showed H accumulation. 2 O 2 accumulation, but the accumulation of OE strain was significantly lower than that of WT ( Figure 7 b). Under control conditions, there was no significant difference in MDA content between WT and OE strains; after 7 days of salt treatment, the MDA content of WT was significantly higher than that of OE strains ( Figure 7 a), indicating that ChtSGAT overexpression can effectively alleviate membrane lipid peroxidation damage induced by salt stress, thereby enhancing the salt tolerance of transgenic Arabidopsis.

[0033] In terms of antioxidant enzyme activity, under control conditions, the superoxide dismutase (SOD) activity of the OE strain was significantly higher than that of the WT, while there was no significant difference in the activities of catalase (CAT) and peroxidase (POD). After salt treatment, the enzyme activities of all plants increased, but the SOD, CAT, and POD activities of the OE strain were significantly higher than those of the WT ( Figure 7 ce).

[0034] The determination of osmotic regulating substances showed that under the control condition, the contents of soluble sugar (SS) and soluble protein (SP) in OE strain were significantly higher than those in WT, while the content of proline (Pro) had no significant difference; after salt stress, osmotic regulating substances of all plants accumulated significantly, and the contents of SS, SP and Pro in OE strain were significantly higher than those in WT ( Figure 7 fh).

Claims

1. A Tokyo ocher SGAT The application of a gene in improving the salt tolerance of a plant is characterized in that: The SGAT The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: SGAT The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that: For amplification SGAT The primers for the gene, the forward primer sequence SEQ ID NO.3 is ChtSGAT-F: 5'-AAAGGTGTTCAGGATAGGGC-3', and the reverse primer sequence SEQ ID NO.4 is ChtSGAT-R: 5'-AATGGTGTTCTGTAGGTACGC-3'.

4. The use according to claim 1, characterized in that: Tokyo tetrapod SGAT Application of genes in regulating salt tolerance of target plants.

5. The use according to claim 4, characterized in that: Tokyo tetrapod SGAT The enhancing effect of gene overexpression on salt tolerance of target plants.

6. The use according to any one of claims 1 to 5, characterized in that: The application is: SGAT The gene is introduced into the target plant, and the resulting transgenic plant has better salt tolerance than the target plant.

7. The use according to any one of claims 1 to 6, characterized in that: The plant is Arabidopsis thaliana.

8. A plant containing the plant represented by SEQ ID NO.1 SGAT Recombinant gene vector.

Images