Tokyo yeshiva Chtsgat gene and its application in improving plant salt tolerance

Abstract

This invention discloses a Tokyo dogwood. ChtSGAT The application of genes in improving plant salt tolerance belongs to the field of plant genetic engineering technology. ChtSGAT The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.2. This invention screened candidate genes for salt tolerance mainly involved in the photosynthetic pathway by analyzing the phenotypic growth, stress physiology, and transcriptomic response of *Cornus kohlii* under salt stress, and also investigated key genes involved in photorespiration. ChtSGAT The results of Arabidopsis thaliana transgenic function verification showed that... ChtSGAT The gene was significantly correlated with the salt tolerance of transgenic Arabidopsis thaliana, improving its salt tolerance. This invention provides a theoretical basis and related genes for breeding salt-tolerant plants.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the Tokyo dogwood. ChtSGAT Genes and their application in improving plant salt tolerance. Background Technology

[0002] Tokyo Four Seasons ( Cornus hongkongensis subsp. tonkinensis This plant belongs to the Cornaceae family and is mainly distributed in China, Japan, and Vietnam. It exhibits strong salt tolerance and diverse leaf colors in coastal saline-alkali lands. Currently, no research has clearly defined its specific characteristics. ChtSGAT The molecular function of the gene in salt tolerance of woody plants is unknown, and there is a lack of genetic transformation and functional verification of the gene. Summary of the Invention

[0003] To address the aforementioned problems, this invention provides the first clone of *Cornus kosoides* from Tokyo. ChtSGAT The gene was constructed using an overexpression vector and its function was verified in transgenic Arabidopsis thaliana. The results showed that this gene can enhance plant salt tolerance through a photorespiration-proline metabolism synergistic pathway. This invention provides genetic resources for developing saline-alkali land-adapted plants that possess both stress resistance and ornamental value.

[0004] This invention provides a Tokyo dogwood. ChtSGAT Application of genes in improving plant salt tolerance.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a ChtSGAT gene from *Cornus kohlii*, the nucleotide sequence of which is shown in SEQ ID NO. 1, specifically as follows:

[0007]

[0008] Furthermore, the present invention provides a protein encoded by the ChtSGAT gene, the amino acid sequence of which is shown in SEQ ID NO.2, specifically as follows:

[0009] MDYVNGPGRNHLFVPGPVNIPEQVIRAMNRNNEDYRSPAIPAMTRTLLEDVKKIFKTTSGTPFLIPTTGTGAWESALTNTLSPGDRIVSFLIGQFSLLWIDQQQRLNFNVDVVESDWGQGANLEVLASKLAADTAHTIKAICIVHNETATGVTNNLSAVRKILDDYRHPALFLVDGVSSICALDFRMDEWGIDVALTGSQK ALLSLPTGMGIVCASPKALEASQTAKSVRVFFDWKDYLKFYKMGTFWPYTPSIQLLYGLRTALDLIFEEGLDNVIARHSRLGKATRLAVEAWGLKNCTQKEEWFSNTVTAVVVPPYIDSSEIVRRGWQRYNLSLGLGLNKVAGKVFRIGHLGNLNDLQLLGCLAGVEMILKDVGYPVKLGSGVAAACAYLQNTIPMIPSRI.

[0010] Secondly, the present invention provides a primer pair for amplifying the above-mentioned ChtSGAT gene, wherein 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'.

[0011] Thirdly, the present invention provides a recombinant vector containing the ChtSGAT gene.

[0012] Fourthly, this invention provides an application of the ChtSGAT gene in regulating the salt tolerance of a target plant.

[0013] Furthermore, the above application specifically demonstrates the effect of overexpression of the ChtSGAT gene in *Cornus kohlii* on enhancing the salt tolerance of the target plant.

[0014] Furthermore, the ChtSGAT gene of *Cornus kohlii* was introduced into the target plant, and the resulting transgenic plant showed better salt tolerance than the target plant.

[0015] Furthermore, the plant in question is Arabidopsis thaliana.

[0016] The beneficial effects of this invention are as follows:

[0017] This invention is the first to clone from Tokyo dogwood. ChtSGAT The gene was constructed using an overexpression vector and its function was verified in transgenic Arabidopsis thaliana. The transgenic Arabidopsis showed significantly improved salt tolerance. Based on these findings, the aforementioned... ChtSGAT Genes can be applied to the genetic improvement of salt tolerance in agricultural and forestry plants or to the breeding of plant varieties with stronger salt tolerance. Attached Figure Description

[0018] Figure 1 In plants SGAT Multiple sequence alignment;

[0019] Figure 2 Phylogenetic tree construction for the ChtSGAT protein sequence;

[0020] Figure 3 Results of hydrophilicity / hydrophobicity analysis of ChtSGAT protein;

[0021] Figure 4 The results of the prediction of the conserved domain of the ChtSGAT protein;

[0022] Figure 5 for ChtSGAT Effects of overexpression on germination and root length of transgenic Arabidopsis thaliana. (ac) represents the germination index, germination rate, and phenotype of Arabidopsis thaliana cultured in MS medium containing 0%, 0.3%, 0.5%, and 0.7% salt for 8 days; (de) represents the root length analysis of seedlings transferred from 3-day-old germination seedlings to saline medium for 8 days. Data are presented as mean ± standard deviation (n=5); the same letter in the bar chart indicates no significant difference between groups (P<0.05).

[0023] Figure 6 Salt stress ChtSGAT Effects of transgenic Arabidopsis thaliana phenotype on chlorophyll fluorescence parameters. (a) Plant phenotype; (b) Chlorophyll fluorescence parameters (Fv / Fm, Fv / F0). Data are presented as mean ± standard deviation (n = 3 biological replicates, 10 seedlings per replicate); error bars represent mean ± standard deviation; *, **, and *** indicate significant differences between WT and transgenic lines under the same treatment conditions (P < 0.05, 0.01, 0.001, respectively). The same applies to the following figures;

[0024] Figure 7 Salt stress ChtSGAT Effects of transgenic Arabidopsis thaliana on physiological responses. (a) MDA content; (b) H2O2 accumulation; (ce) antioxidant enzyme activity (SOD, CAT, POD); (fh) osmotic regulator content (SS, SP, Pro). Detailed Implementation

[0025] To facilitate a better understanding of the present invention, the following description will further illustrate the invention with reference to some embodiments. However, the scope of protection of the present invention is not limited to the following embodiments. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the invention are all within the scope of the present invention. Test methods in the following embodiments that do not specify specific conditions are all conventional methods.

[0026] Plant materials and growing conditions

[0027] The test plants were one-year-old seedlings of *Cornus kohlii*. Seedlings with similar growth and good condition were selected and transplanted into 50 L hydroponic incubators (length × width × height: 110 cm × 57 cm × 16 cm). 50 L of 1 / 2 Hoagland modified nutrient solution was added to the hydroponic incubator, aeration was provided for 24 hours, the pH was set to 6.0, the nutrient solution was changed every 7 days, and an aeration system was installed to ensure adequate dissolved oxygen.

[0028] Seeds of Arabidopsis ecotype Columbia were surface-sterilized with 10% sodium hypochlorite for 20 min, followed by washing three times with sterile water. The sterilized seeds were inoculated onto 1 / 2 MS medium and stored at 4°C for 3 days before germination. Seven-day-old seedlings were then transferred to a mixture of nutrient-rich soil and perlite (2:1) and grown at 23°C under a light intensity of 200 μmol / m². −2 s −1 The light cycle is 16 / 8 h (light / dark).

[0029] Example 1: ChtSGAT Gene cloning and analysis

[0030] 1. ChtSGAT Cloning of genes

[0031] Total RNA was extracted from young leaves of *Cornus kohlii* using the RNA prep Pure Plant Kit (Tiangen, China). RNA concentration and quality were determined using a NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and 1% agarose gel electrophoresis, respectively. First-strand cDNA was synthesized from the total RNA using the PrimeScript™ II First-Strand cDNA Synthesis Kit (Takara, Japan). Full-length primers were designed based on the transcriptome assembly sequence. The full-length primer sequences are as follows:

[0032] ChtSGAT-F:

[0033] 5'-AAAGGTGTTCAGGATAGGGC-3' (SEQ ID NO.3)

[0034] ChtSGAT-R:

[0035] 5'-AATGGTGTTCTGTAGGTACGC-3' (SEQ ID NO.4)

[0036] (1) Full-length sequence amplification

[0037] A high-fidelity enzyme amplification system was used to obtain the full-length fragment. The PCR system is as follows:

[0038] 5 PrimeSTAR Buffer (Mg 2+ Plus) 10 μL

[0039] dNTP Mixture (2.5 mM each) 4 μL

[0040] Primer F 1 μL

[0041] Primer R 1 μL

[0042] cDNA <200 ng

[0043] PrimeSTAR HS DNA Polymerase 0.5 μL

[0044] ddH2O up to 50 μL

[0045] 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; storage at 4°C.

[0046] (2) Gel recovery of PCR products

[0047] PCR products were recovered by gel extraction using a 1% agarose gel electrophoresis kit (Axygen, USA), following the kit's instructions. Vector ligation was performed using the pClone007 SimpleVector Kit from Qingke Biotechnology.

[0048] (3) Competency transformation

[0049] Ice-thawed E. coli competent cells (Trans Chemically Competent Cell, Beijing Jinshi Column): Add 10 μL of ligation buffer to a final volume of 100 μL of competent cells. Incubate on ice for 30 min → heat shock at 42°C for 90 s → incubate on ice for 2 min → add 800 μL of antibiotic-free LB medium and incubate at 37°C and 220 rpm for 0.5–1 h. Centrifuge at 4000 rpm for 3 min at room temperature, resuspend in 100 μL of the bacterial culture, spread on Amp+ (100 μg / mL) plates, and incubate upside down at 37°C for 12–16 h.

[0050] (4) Screening and detection of monoclonal bacteria

[0051] Pick a single white colony with a pipette tip and add it to 10 μL of sterile water. Mix well by pipetting, then add 1 μL to 20 μL of Super PCRMix (green) and stir gently. Amplify according to the pClone007 Simple Vector Kit program. Send the PCR product of positive bacteria to Nanjing Qingke Biotechnology for sequencing.

[0052] 2. Obtaining the complete ORF

[0053] The obtained sequence will be concatenated using the CAP3 Sequence Assembly Program (http: / / doua.prabi.fr / software / cap3) to obtain a complete sequence.

[0054] 3. ChtSGAT Gene sequence analysis

[0055] ChtSGAT The full-length ORF of the gene is 1206 bp, and the protein it encodes consists of 401 amino acids with a molecular weight of 44.09 kDa and an isoelectric point of 8.13.

[0056] Will ChtSGAT The nucleotide sequence of the gene was subjected to NCBI-Blast alignment analysis to explore its phylogenetic relationship with other species. Representative species with high homology were selected from the NCBI database for amino acid sequence homology alignment analysis. The analysis results were performed using Genedoc software, and molecular phylogenetic trees among different species were constructed using mega software. The results are as follows: Figure 1 and Figure 2 As shown, the results are... ChtSGAT Because of their close kinship with other species, SGAT is presumably derived from a common ancestor. ChtSGAT With blueberry tree ( Nyssa sinensis (KAA8549846.1) and tea ( Camellia sinensisThe sequence (XP_028060239.1) showed the highest homology, with a similarity of 94.76%.

[0057] In the ExPASy online webpage, select the ProtScale analysis software. Paste the sequence on the ProtScale homepage and select to analyze. This will provide the results of the protein's hydrophilicity and hydrophobicity analysis. If the hydrophobic regions are more numerous than the hydrophilic regions overall, it indicates that the protein is more hydrophobic than hydrophilic, and may be classified as a hydrophobic protein. Figure 3 ).

[0058] Through conservative structure and predictive analysis of SGAT, the results are as follows: Figure 4 As shown. ChtSGAT The gene encodes a protein with a conserved structure of serine-glyoxylate aminotransferase and is the only member of the superfamily cl30752. It contains three conserved sites found in terpene synthases.

[0059] Example 2: ChtSGAT Gene function identification

[0060] 1. Construction of transgenic plants

[0061] Will ChtSGAT The gene sequence was cloned into the pCAMBIA 2300-eGFP vector driven by the 35S:eGFP promoter. Wild-type Arabidopsis thaliana (Col-0) was transformed with Agrobacterium tumefaciens GV3101 carrying the recombinant plasmid using a modified inflorescence immersion method. Positive lines were screened on 1 / 2 MS medium containing 50 mg / L kanamycin, and after PCR verification, selection was performed by qRT-PCR. ChtSGAT The third-generation homozygous transgenic lines with overexpressed genes were used for subsequent experiments.

[0062] 2. Salt tolerance phenotypic analysis

[0063] Three transgenic lines ( ChtSGAT -OE1、 ChtSGAT -OE2、 ChtSGAT -OE3) and wild-type (WT) Arabidopsis thaliana seeds were placed on 1 / 2 MS medium containing 0%, 0.3%, 0.5%, and 0.7% salt, respectively. After vernalization at 4°C for 3 days, they were germinated in a 22°C light incubator. Seed germination parameters were recorded over 24 hours and observed continuously for 8 days. Germination rate (GR) and germination index (GI) were calculated.

[0064] GR = (Number of germinating seeds in each treatment / Total number of seeds) × 100%

[0065] GI = Σ (Number of seeds germinating on day t / Corresponding number of germination days)

[0066] Seedlings that had germinated for 3 days were transferred to fresh culture medium with the same salt concentration, and root length was measured 8 days later. WT and transgenic plants that had grown for 4 weeks were also taken and irrigated with a 0.5% salt solution for 3 consecutive days. After 7 days, Fv / Fm and Fv / F0 were measured using Handy PEA. Simultaneously, leaves were collected to measure H2O2 and MDA content, and the activities of SOD, CAT, and POD, as well as the content of osmotic regulators (SS, SP, Pro). H2O2 accumulation was detected by staining with 3,3'-diaminobenzidine (DAB, CAS: 7411-49-6) (Bapatla et al., 2021), and peroxidase (POD) activity was measured according to the method of Yin et al. (2018).

[0067] 3. ChtSGAT Gene overexpression enhances salt tolerance in transgenic Arabidopsis thaliana

[0068] Analysis of the germination characteristics of ChtSGAT overexpression lines (OE1, OE2, OE3) and wild-type (WT) under salt stress revealed that germination of both WT and transgenic lines was inhibited with increasing salt concentration. Figure 5 a), but the germination index of the transgenic lines was consistently significantly higher than that of WT ( Figure 5 b). Under salt-free conditions, the transgenic lines showed a higher germination rate than WT lines only on day 2; at salt concentrations of 0.3% and 0.5%, the transgenic lines exhibited a significant advantage in the early stages of germination (days 1-2) (P<0.05). Figure 5 c) indicates that ChtSGAT overexpression may accelerate the early germination process of seeds. When the salt concentration reaches 0.7%, the germination rate of the transgenic lines is significantly higher than that of WT from the 3rd day of germination. Figure 5 c). However, there was no significant difference in root length between WT and transgenic lines at different salt concentrations ( Figure 5 de).

[0069] Observations on 4-week-old WT and OE seedlings treated with 0.5% salt solution revealed that the growth of both WT and OE seedlings was inhibited with increasing salt treatment duration, but the overall growth of the OE seedlings was better than that of the WT seedlings. Figure 6 a). Under control conditions (CK), there were no significant differences in Fv / Fm (maximum photochemical efficiency) and Fv / F0 (potential photosystem II activity) between OE and WT; however, under 0.5% salt stress, the Fv / Fm and Fv / F0 values ​​of the OE line were significantly higher than those of WT (P<0.05), indicating that its photosynthetic system stability was significantly enhanced. Figure 6 b).

[0070] Under salt-free conditions, no obvious DAB staining signal (H2O2 accumulation) was detected in either WT or transgenic seedlings, and there was no significant difference between groups; however, under 0.5% salt stress, although all plants showed H2O2 accumulation, the accumulation in the OE line was significantly lower than that in the WT line. Figure 7 b). Under control conditions, there was no significant difference in MDA content between the WT and OE lines; after 7 days of salt treatment, the MDA content of the WT line was significantly higher than that of the OE line. Figure 7 a) indicates that ChtSGAT overexpression can effectively alleviate membrane lipid peroxidation damage caused by salt stress, thereby enhancing the salt tolerance of transgenic Arabidopsis thaliana.

[0071] Regarding antioxidant enzyme activity, under control conditions, the superoxide dismutase (SOD) activity of the OE strain was significantly higher than that of the WT strain, while there was no significant difference in catalase (CAT) and peroxidase (POD) activities. Salt treatment increased enzyme activities in all plants, but the SOD, CAT, and POD activities of the OE strain were significantly higher than those of the WT strain. Figure 7 ce).

[0072] Osmotic regulation substance determination showed that, under control conditions, the contents of soluble sugar (SS) and soluble protein (SP) in the OE line were significantly higher than those in the WT line, while the content of proline (Pro) showed no significant difference. After salt stress, all plants showed significant accumulation of osmotic regulation substances, and the contents of SS, SP, and Pro in the OE line were significantly higher than those in the WT line. Figure 7 fh).

Claims

1. A type of Tokyo dogwood ChtSGAT The application of genes in improving salt tolerance in Arabidopsis thaliana is characterized by, The aforementioned ChtSGAT The nucleotide sequence of the gene is shown in SEQ ID NO.

1. ChtSGAT The protein amino acid sequence of the gene is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, For amplification ChtSGAT Gene primers: The forward primer sequence SEQ ID NO.3 is ChtSGAT-F: 5'-AAAGGTGTTCAGGATAGGGC-3'; The reverse primer sequence SEQ ID NO.4 is ChtSGAT-R: 5'-AATGGTGTTCTGTAGGTACGC-3'.

3. The application according to claim 1, characterized in that, The Tokyo Four Seasons Flower ChtSGAT The effect of gene overexpression on enhancing salt tolerance in Arabidopsis thaliana.

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