Safflower CtGSTF3 gene and application thereof in regulating anthocyanin synthesis and improving drought and salt stress resistance of plants

Abstract

The application discloses glutathione S-transferase gene CtGSTF3 derived from safflower and application of the gene in positive regulation of anthocyanin accumulation of plants and improvement of drought and salt stress resistance of plants. The full-length nucleotide sequence of the CtGSTF3 gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2. The application first obtains the complete coding sequence of CtGSTF3 by RT-PCR cloning, and completes the heterologous function verification in Arabidopsis tt19 mutants. The results show that overexpression of CtGSTF3 can restore the anthocyanin synthesis defect phenotype of tt19 mutants, up-regulate the expression of anthocyanin synthesis related genes, promote the accumulation of anthocyanin, and prove that CtGSTF3 can positively regulate the biosynthesis of anthocyanin of plants. Meanwhile, the heterologous expression of CtGSTF3 can alleviate the sensitivity of tt19 mutants to drought and salt stress, and significantly improve the stress tolerance of plants. In conclusion, the CtGSTF3 gene can be used for plant color improvement and new variety cultivation, and provides a new gene resource for plant stress resistance breeding, and has a wide application prospect in the creation of new varieties of drought and saline-alkali tolerant plants.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of a safflower CtGSTF3 protein and its gene in regulating anthocyanin synthesis and resistance to drought and salt stress. Background Technology

[0002] Anthocyanins are a class of water-soluble flavonoid plant secondary metabolites, mostly accumulated in the vacuoles of plant epidermal cells in the form of glycosides. They are key pigments determining the coloring of petals, fruits, and vegetative organs. Anthocyanins possess significant antioxidant activity, mediating not only plant responses to various biotic and abiotic stresses but also providing numerous beneficial effects on human health. Anthocyanins are biosynthesized on the endoplasmic reticulum membrane and subsequently transported into the vacuoles for storage. The glutathione S-transferase (GST) family is the core functional gene family of the anthocyanin transport pathway. Through the glutathione binding escort mechanism, it drives the efficient transport of anthocyanins to vacuoles for accumulation, deeply participating in the plant pigment formation process and is an important gene family regulating plant coloring.

[0003] Glutathione S-transferase (GST) belongs to the multifunctional enzyme family and is widely involved in the regulation of plant growth and development and responses to abiotic stress. GST was first discovered in the 1960s in animal drug detoxification research; in the 1980s, researchers first identified plant GST in herbicide-treated maize. Plant GST mainly functions as a detoxification agent, catalyzing the binding of glutathione (γ-Glu-Cys-Gly) with hydrophobic and electrophilic compounds to generate S-glutathione derivatives. Based on sequence characteristics, plant soluble GST can be divided into several subfamilies, including GSTU (tau), GSTF (phi), GSTT (theta), GSTZ (zeta), GSTL (lambda), DHAR (dehydroascorbic acid reductase), and TCHQD (tetrachlorohydroquinone dehalogenase). Existing research confirms that Phi-type GSTs are core members involved in the transport of secondary metabolites and stress responses in plants, such as maize Bz2, Arabidopsis thaliana TT19, and petunia An9. These proteins can promote the transport of anthocyanins to vacuoles for accumulation through glutathione binding or molecular chaperone escort mechanisms, preventing their oxidative degradation in the cytoplasm. Simultaneously, GSTs participate in the construction of plant antioxidant defense systems, enhancing plant tolerance to adversity by scavenging reactive oxygen species and detoxifying harmful substances.

[0004] Safflower (Carthamus tinctorius L.) is an annual herbaceous plant belonging to the Asteraceae family. It is a traditional medicinal plant cultivated in my country for over two thousand years. Different genotypes of safflower can produce a rich variety of flower colors, including red, orange, yellow, and white, possessing diverse economic value and potential for development as a medicinal raw material, natural pigment resource, and ornamental plant. Dried safflower flowers are commonly used in traditional Chinese medicine, possessing antioxidant and blood-activating activities, and are widely used in the prevention and treatment of cardiovascular and cerebrovascular diseases. Phenolic and flavonoid secondary metabolites are important pharmacodynamic substances. Safflower exhibits a certain degree of tolerance to various environmental stresses such as drought, salinity, and extreme temperatures, with a wide range of cultivation adaptability and significant potential for discovering stress-resistance genes. Discovering functional genes for stress response in safflower and elucidating its molecular mechanisms of stress adaptation can not only improve the theoretical system of plant stress resistance regulation but also provide genetic resources and breeding materials for the selection of new safflower varieties suitable for arid and saline-alkali areas.

[0005] Currently, GST genes that regulate anthocyanin accumulation have been identified in plants such as apples, peaches, and cotton. However, research on the anthocyanin metabolism regulation and stress resistance mechanisms of safflower, an important medicinal and oilseed crop, lags behind. There are no reports on the cloning, functional identification, or application of safflower GST genes, which hinders safflower variety improvement and anthocyanin resource development. Therefore, cloning and identifying CtGST genes from safflower that simultaneously regulate anthocyanin synthesis and stress resistance, elucidating their mechanism of action, and exploring their application potential are of significant theoretical and practical value for promoting molecular breeding work in safflower and other crops. Summary of the Invention

[0006] The purpose of this invention is to provide a safflower glutathione S-transferase gene CtGSTF3, which can regulate the synthesis of anthocyanins in plants and the resistance to drought and salt stress. This gene can be used to construct transgenic plants with high anthocyanin content and can also be applied to the breeding of new plant varieties with strong stress resistance.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a safflower glutathione S-transferase gene CtGSTF3, the CDS sequence of which is shown in SEQ ID NO.1.

[0009] The present invention also provides a protein encoded by the above-mentioned safflower CtGSTF3 gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0010] This invention also provides the application of the above-mentioned safflower CtGSTF3 gene in regulating anthocyanin synthesis in plants and constructing transgenic plants with high anthocyanin content.

[0011] Preferably, the plant includes Arabidopsis thaliana.

[0012] This invention also provides the application of the above-mentioned safflower CtGSTF3 gene in regulating plant resistance to drought and salt stress and constructing drought- and salt-tolerant transgenic plants.

[0013] The present invention also provides a method for increasing the anthocyanin content and stress resistance of plants, wherein the method comprises: overexpressing the safflower CtGSTF3 gene in the plant.

[0014] Preferably, the overexpression is achieved by introducing a recombinant expression vector carrying the safflower CtGSTF3 gene into the target plant.

[0015] Preferably, the recombinant vector is a plant overexpression vector containing the CtGSTF3 gene.

[0016] The present invention also provides the application of recombinant vectors containing the above-mentioned safflower CtGSTF3 gene in improving anthocyanin content and stress resistance in plants.

[0017] This invention also provides the application of the above-mentioned safflower CtGSTF3 gene and its recombinant vector in plant color improvement and stress resistance genetic engineering breeding.

[0018] Overexpression of the safflower CtGSTF3 gene in plants significantly promotes anthocyanin accumulation and deepens plant tissue coloration; it also significantly enhances the plant's tolerance to drought and salt stress. Specifically, this enhanced tolerance manifests as promoting normal plant growth and development under stress conditions and increasing the activities of superoxide dismutase, peroxidase, and catalase in the plant.

[0019] The beneficial effects of this invention are:

[0020] This invention utilizes RT-PCR technology to clone the full-length CDS sequence of the safflower glutathione S-transferase gene CtGSTF3. Functional verification results show that CtGSTF3 has a dual function of regulating anthocyanin synthesis and drought and salt stress resistance in plants. Heterologous overexpression of CtGSTF3 in the Arabidopsis tt19 mutant can restore the mutant's anthocyanin synthesis defect phenotype. Therefore, this gene can be used to create transgenic materials with high anthocyanin content and applied to the breeding of new plant varieties for color improvement. Simultaneously, overexpression of CtGSTF3 in the Arabidopsis tt19 mutant can significantly enhance the plant's resistance to drought and salt stress, providing a novel gene target for molecular breeding of plant stress resistance and showing promising application prospects in the field of drought- and salt-tolerant crop cultivation. Attached Figure Description

[0021] Figure 1 shows the sequence characteristics and gene expression patterns of CtGSTF3. (A) Multiple sequence alignment results of CtGSTF3 with GST proteins from other species; (B) Phylogenetic tree of CtGSTF3 with GST proteins from other species; (C) Expression patterns of CtGSTF3 under drought and salt stress treatments; different lowercase letters indicate significant differences between groups (P < 0.05).

[0022] Figure 2 shows the functional complementation verification of CtGSTF3 in the Arabidopsis thaliana tt19 mutant. (A) Plant phenotypes of wild-type (Col), tt19 mutant, and CtGSTF3 / tt19 complementary lines; (B, C) Expression levels of CtGSTF3 gene in wild-type (Col), tt19 mutant, and CtGSTF3 / tt19 complementary lines; (D) Anthocyanin content in wild-type (Col), tt19 mutant, and CtGSTF3 / tt19 complementary lines; (EK) Relative expression levels of anthocyanin synthesis-related genes in wild-type (Col), tt19 mutant, and CtGSTF3 / tt19 complementary lines; bar = 5 mm in the figure, and different lowercase letters indicate significant differences between groups (P < 0.05).

[0023] Figure 3 shows the subcellular localization analysis of CtGSTF3 protein and the functional verification of CtGSTF3 in yeast under drought and salt stress. (A) Observation results of subcellular localization fluorescence of CtGSTF3 in tobacco leaf epidermal cells; (B) Observation results of subcellular localization fluorescence of CtGSTF3 in maize protoplasts; (C) Maps of empty vector pYES2 and recombinant vector pYES2-CtGSTF3; (D) Growth of yeast strains carrying empty pYES2 vector and pYES2-CtGSTF3 after 5 days of culture in SGR medium supplemented with different concentrations of NaCl (0, 1.5 M, 2 M) or mannitol (0, 0.5 M, 1.5 M).

[0024] Figure 4 shows the functional verification of CtGSTF3 enhancing the resistance of Arabidopsis thaliana to drought and salt stress. (A) Phenotypes of wild-type (Col), tt19 mutant, and CtGSTF3 / tt19 complementary lines after treatment with drought and salt stress; (B) DAB and NBT staining results of wild-type (Col), tt19 mutant, and CtGSTF3 / tt19 complementary lines after treatment with normal conditions, drought stress, and salt stress; (CE) CAT, POD, and SOD enzyme activity detection results of wild-type (Col), tt19 mutant, and CtGSTF3 / tt19 complementary lines after treatment with drought and salt stress; in the figure, bar = 1 cm, and different lowercase letters indicate significant differences between groups (P < 0.05).

[0025] Figure 5 shows the expression levels of anthocyanin synthesis-related genes and the anthocyanin content of plants under different treatment conditions. (AG) Relative expression levels of anthocyanin synthesis-related genes in wild-type (Col), tt19 mutant, and CtGSTF3 / tt19 complementary lines under normal conditions, drought stress, and salt stress treatment; (H, I) Anthocyanin content in wild-type (Col), tt19 mutant, and CtGSTF3 / tt19 complementary lines under normal conditions, drought stress, and salt stress treatment; bar = 5 mm in the figure, and different lowercase letters indicate significant differences between groups (P < 0.05).

[0026] Figure 6 shows the results of stress response gene expression detection in plants after stress treatment. The relative expression levels of stress response genes in wild-type (Col), tt19 mutant, and CtGSTF3 / tt19 complementary lines after normal conditions, drought stress, and salt stress treatment; different lowercase letters indicate significant differences between groups (P < 0.05). Detailed Implementation The present invention will now be described in more detail with reference to the accompanying drawings. The following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional molecular biology procedures, and specific procedures can be found in *Molecular Cloning: A Laboratory Manual* (3rd edition). Unless otherwise specified, the reagents used in the embodiments were purchased from conventional biochemical reagent suppliers; unless otherwise specified, the instruments and equipment used are conventional laboratory instruments. All quantitative experiments in the following embodiments were performed in triplicate, and the results were averaged.

[0027] Example 1 Cloning of the CtGSTF3 gene in safflower

[0028] Using the Unigene sequence of the safflower transcriptome constructed from the germplasm resource bank of the Institute of Chinese Medicinal Herbs, Henan Academy of Agricultural Sciences as a reference sequence, full-length amplification primers were designed and synthesized by Henan Youkang Gene Biotechnology Co., Ltd.: upstream primer CtGSTF3-F (5′-ATGGTGGTCAAGGTGTATGGTC-3′, SEQ ID NO.3) and downstream primer CtGSTF3-R (5′-ACTCTGTTAGTCGTGTGACCTT-3′, SEQ ID NO.4).

[0029] Total RNA was extracted from safflower using the Nanjing Novizan FastPure® Universal Plant Total RNA Isolation Kit, and cDNA was obtained by reverse transcription of the total RNA using the HiScript® III 1st Strand cDNA Synthesis Kit (+gDNA wiper).

[0030] Using safflower cDNA as a template, the complete CDS sequence of CtGSTF3 was amplified using Toyobo KOD One high-fidelity DNA polymerase (Toyobo, Japan). The amplified product was purified using a TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver. 4.0 (TaKaRa, Japan). The purified product was ligated into a T vector and transformed into DH5α competent cells. Single colonies were picked for PCR identification and sequencing verification. The sequencing results showed that the obtained full-length CDS sequence of the CtGSTF3 gene was 642 bp, and the sequence information is shown in SEQ ID NO.1.

[0031] Example 2: Sequence Feature Analysis of the CtGSTF3 Gene in Safflower

[0032] The CtGSTF3 gene has an open reading frame of 642 bp, encoding a polypeptide of 213 amino acids, as shown in SEQ ID NO.2. Multiple sequence alignment (MSA) results showed that CtGSTF3 shared 52.34%, 61.64%, 68.08%, and 69.77% amino acid sequence homology with Arabidopsis thaliana AtTT19, petunia PhAN9, grape VvGSTF12, and apple MdGSTF6, respectively (Figure 1A). Phylogenetic analysis revealed that CtGSTF3 clustered with AtTT19, VvGSTF12, PsGSTF3, and FvRAP (Figure 1B). Previous studies have confirmed that AtTT19 is responsible for anthocyanin transport to the vacuoles; PsGSTF3 participates in anthocyanin transport but not proanthocyanidin transport. Based on this, it is speculated that CtGSTF3 is a candidate protein for anthocyanin transport regulating safflower coloration.

[0033] Example 3: Analysis of the expression pattern of the CtGSTF3 gene under abiotic stress

[0034] Select plump and uniform-sized seeds of Yuhonghua No. 4 and sow them in vermiculite substrate. Six days after germination, remove the seedlings, rinse the roots with water, and transplant them into Hoagland nutrient solution for hydroponic cultivation. After two weeks of hydroponics, select seedlings with uniform growth and transfer them to solutions supplemented with 20% PEG6000 and 100 mmol·L⁻¹ nutrient solution, respectively.-1 Hoagland nutrient solution containing NaCl simulated drought and salt stress treatments. The third functional leaf of the plant was collected at 0 h, 3 h, 12 h, 24 h, and 48 h after treatment. Total RNA was extracted according to the method described in Example 1. Quantitative PCR was performed using the GenStar RealStar Fast SYBR Mix kit. The CtGSTF3 quantitative primers were: qCtGSTF3-F (5′-AGAGTATTGGATTGCCTTTTCG-3′, SEQ ID NO.5) and qCtGSTF3-R (5′-GGGTTGTTTATGCTCGTTGG-3′, SEQ ID NO.6).

[0035] The results are as follows Figure 1 As shown in C: Under drought stress, the expression level of CtGSTF3 gene was significantly upregulated at all time points, reaching a peak at 24 h, which was 14.6 times that of the control group; under salt stress, the expression level of CtGSTF3 increased rapidly within 3 h, reaching a peak at 12 h, which was 375.6 times that of the control group, and then the expression level gradually decreased.

[0036] Example 4 Construction of CtGSTF3 overexpression vector and genetic transformation of Arabidopsis thaliana tt19 mutant

[0037] 4.1 Construction of CtGSTF3 plant overexpression vector

[0038] The 3300B vector was linearized using restriction endonucleases EcoRI and BamHI, and primers carrying homologous recombination arms and restriction sites were designed: 3300B-CtGSTF3-F (5′-acgggggactcttgacGAATTCATGGTGGTCAAGGTGTATGGTC-3′, SEQ ID NO.7) and 3300B-CtGSTF3-R (5′-cgagctggtcaccCTAGGATCCACTCTGTTAGTCGTGTGACCTT-3′, SEQ ID NO.8).

[0039] The complete coding sequence of CtGSTF3 was amplified using Toyobo KOD One high-fidelity enzyme, and the amplified product was purified using a TaKaRa gel extraction kit. The linearized vector and purified PCR fragment were ligated using the Nanjing Novizan Clone Express® II One Step Cloning Kit for homologous recombination. The ligation product was transformed into DH5α competent cells. Positive clones were selected for PCR identification, and recombinant plasmids were extracted. The recombinant vector 35S::CtGSTF3 was sequenced and verified by Henan Youkang Gene Biotechnology Co., Ltd.

[0040] 4.2 Agrobacterium-mediated transformation of Arabidopsis flowers

[0041] (1) Plant cultivation: The seeds of Arabidopsis thaliana tt19, a mutant of anthocyanin transporter GST gene deletion, were inoculated on 1 / 2 MS solid medium for germination. After the seedlings developed their second true leaves, they were transplanted to nutrient soil. They were then cultivated in an artificial climate chamber with 16 h of light / 8 h of darkness, daytime temperature of 24 ℃ / nighttime temperature of 22 ℃, and relative humidity of 50%~60% until the flowering period.

[0042] (2) Preparation of Agrobacterium tumefaciens bacterial culture: Agrobacterium tumefaciens GV3101 carrying the recombinant vector 3300B-35S::CtGSTF3 was inoculated into LB liquid medium containing 50 μg / mL rifampin (Rif) and 50 μg / mL kanamycin (Kan), and cultured with shaking at 28 ℃ and 220 rpm; the activated bacterial culture was then transferred for expansion culture until OD. 600 ≈1.0. Collect bacterial cells by centrifugation at 6000 rpm for 6 min, resuspend the cells in an aqueous solution containing 5% sucrose and 0.03% Silwet L-77, and adjust the OD of the bacterial solution. 600 Up to 0.4.

[0043] (3) Flower inoculation transformation: Remove open flowers and mature siliques of Arabidopsis thaliana, leaving only unpollinated flower buds; immerse the aboveground parts of the plant in Agrobacterium tumefaciens inoculation solution for 30 s, repeating 2-3 times to ensure sufficient inoculation. After inoculation, place the plant horizontally and incubate in the dark and humidified environment for 24 h, then restore normal culture conditions; repeat the inoculation 2-3 times after the first inoculation 3-7 days later to improve transformation efficiency.

[0044] (4) Screening of positive transgenic plants: T1 generation seeds were harvested and evenly sown in nutrient soil, covered with film to retain moisture; after the first pair of true leaves of the seedlings had fully unfolded, a 20 mg / L effective glufosinate solution was sprayed, and the spraying was repeated once every 3-4 days. After 7-10 days of spraying, surviving plants were screened as positive candidates, and wild-type Col was sprayed simultaneously as a negative control. Positive plants were harvested individually and continuously screened up to the T3 generation to obtain genetically stable homozygous complementary lines. Homozygous lines #3 and #8 were selected for subsequent functional verification.

[0045] Example 5: Identification of functional complementarity phenotypes in CtGSTF3 transgenic Arabidopsis thaliana

[0046] 5.1 Molecular identification and phenotypic observation of transgenic plants

[0047] Total RNA was extracted from wild-type Col, tt19 mutant, and transgenic complementary lines #3 and #8 using the RNA extraction method described in Example 1, and cDNA was obtained by reverse transcription. CtGSTF3 was amplified using the cDNA as a template, and the results are as follows. Figure 2 As shown in Figure B: No specific amplification bands were observed in the wild-type and tt19 mutant, but the target band was amplified in the transgenic lines, proving that CtGSTF3 was successfully introduced into the tt19 mutant. qRT-PCR detection was performed using the method described in Example 3.

[0048] Independent lines #3 and #8 with high CtGSTF3 expression were obtained by qRT-PCR screening (Figure 2C). Phenotypic observation results showed that the 35S::CtGSTF3 / tt19 line restored the purple pigment accumulation in the rosette leaf base, leaves, and stems of the wild type, and could complement the color defect phenotype of the tt19 mutant plant; however, the transgenic lines could not restore the seed coat color defect of the tt19 mutant. Figure 2 A). This result indicates that CtGSTF3 is involved in anthocyanin transport in vegetative organs but not in proanthocyanidin transport in the seed coat.

[0049] 5.2 Detection of anthocyanin content and gene expression in the synthesis pathway

[0050] Arabidopsis thaliana leaves were thoroughly ground with liquid nitrogen. 0.1 g of the powder was placed in a 1.5 mL centrifuge tube, and 1 mL of 1% hydrochloric acid-methanol solution was added. Extraction was carried out overnight in the dark. Then, 500 μL of distilled water and 500 μL of chloroform were added sequentially, and the mixture was centrifuged at 12,000 r / min for 10 min at 4℃. 200 μL of the supernatant was collected, and the absorbance at 530 nm was measured using a Varioskan LUX microplate reader (Thermo Fisher Scientific). The anthocyanin content was calculated using the formula: C (mg / g) = (A... 530 (×MW×DF×V) / (ε×m×l) Where: C is the anthocyanin content (mg / g); A 530 530 nm absorbance of the sample; MW is the molar mass of cyanidin-3-O-glucoside (449.2 g / mol); DF is the dilution factor; V is the total volume of the extract (mL); ε is the molar extinction coefficient of cyanidin-3-O-glucoside (29,600 L·mol⁻¹). -1 ·cm -1 m is the sample mass (g); L is the optical path length (1 cm).

[0051] Quantitative results such as Figure 2 As shown in Figure D, the anthocyanin content in transgenic lines #3 and #8 was approximately 2.2 times that of the tt19 mutant; seven key structural genes in the anthocyanin biosynthesis pathway were significantly upregulated in the transgenic lines (Figure 2E-K). These results confirm that CtGSTF3 mediates anthocyanin transport and accumulation.

[0052] Example 6: Subcellular localization analysis of CtGSTF3 protein

[0053] The pSUPER1300-EGFP vector was linearized using Hind III and Kpn I; homologous recombination primers were designed: EGFP-CtGSTF3-F (5′-CAAATCGACTCTAGAAAGCTTATGGTGGTCAAGGTGTATGGTC-3′, SEQ ID NO.9) and EGFP-CtGSTF3-R (5′-GCCCTTGCTCACCATGGTACCCTCTGTTAGTCGTGTGACCTT-3′, SEQ ID NO.10).

[0054] The CtGSTF3 coding sequence with the stop codon removed was amplified using KOD One high-fidelity enzyme, homologously recombinated and ligated into a linearized vector to construct a 35S::CtGSTF3-GFP fusion expression vector. The vector construction method was the same as in Example 4.

[0055] The recombinant vector 35S::CtGSTF3-GFP and the empty vector plasmid were transformed into Agrobacterium GV3101, respectively; positive strains were cultured at OD200 by LB culture. 600 ≈1.0, bacterial cells were resuspended using resuspension buffer (10 mM MgCl2, 10 mM MES, 200 μM AS) and OD was adjusted. 600 To 1.0, the abaxial surface of 4-week-old tobacco leaves was injected and cultured normally for 48 h before observing fluorescence. Maize protoplasts were prepared using the PEG-calc-mediated method and transfected with plasmids: 10 μg of plasmid and 100 μL of protoplasts (4 × 10⁻⁶) were used. 5 Mix well with PEG transfection buffer (40% w / v PEG 4000, 0.6 M mannitol, 100 mM CaCl2), and incubate at 25 °C in the dark for 15 min. Stop transfection by adding 440 μL of W5 solution (150 mM NaCl, 100 mM CaCl2, 0.5 mM KH2PO4, 0.5 mM MES, pH 4.8), and incubate overnight at 25 °C in the dark. Observe the GFP fluorescence signal using a Zeiss ZESIS 710 laser confocal microscope.

[0056] Fluorescence observation results as follows Figure 3 As shown in A and 3B: the empty GFP green fluorescence is diffusely distributed in the cytoplasm, cell membrane and cell nucleus; the fluorescence signal of the 35S::CtGSTF3-GFP fusion protein is localized in the cell membrane and cell nucleus, proving that CtGSTF3 is a dual-localized protein in the cell membrane and cell nucleus.

[0057] Example 7: Heterologous expression of CtGSTF3 enhances yeast tolerance to drought and salt stress

[0058] 7.1 Construction of pYES2-CtGSTF3 yeast expression vector

[0059] The pYES2 vector was linearized using EcoRI and XhoI; homologous recombination primers were designed: pYES2-CtGSTF3-F (5′-GCCGCCAGTGTGCTGGAATTCATGGTGGTCAAGGTGTATGGTC-3′, SEQ ID NO.11) and pYES2-CtGSTF3-R (5′-CCCTCTAGATGCATGCTCGAGACTCTGTTAGTCGTGTGACCTT-3′, SEQ ID NO.12).

[0060] The complete coding sequence of CtGSTF3 was amplified and homologously recombinated into the linearized pYES2 vector to construct the pYES2-CtGSTF3 recombinant vector. The construction method is as described in Example 4.

[0061] 7.2 Validation of yeast function under stress

[0062] The recombinant plasmid pYES2-CtGSTF3 and the empty vector pYES2 were transformed into competent Saccharomyces cerevisiae BY4741 cells, respectively, and plated on SD / -Ura solid medium. The cells were incubated at 30 °C for 3–5 days. Positive single colonies were picked, resuspended in sterile water, and the OD was uniformly adjusted. 600 ; Perform gradient dilution sequentially to obtain OD 600 10 0 10 -1 10 -2 10 -3 Bacterial suspensions were prepared. 3 μL of the bacterial suspension was spot-spread onto SG-Ura plates containing different concentrations of NaCl (0, 1.5 M, 2 M) and mannitol (0, 0.5 M, 1.5 M) and incubated at 30 ℃ for 5 days.

[0063] Growth results as follows Figure 3 As shown in Figure D, yeast growth was inhibited with increasing concentrations of NaCl and mannitol. Under the same stress conditions, the yeast strain expressing pYES2-CtGSTF3 showed significantly better growth than the empty pYES2 strain, demonstrating that CtGSTF3 can enhance the yeast's tolerance to drought-simulated stress and salt stress.

[0064] Example 8: Drought and Salt Stress Treatments and Phenotypic Identification in Transgenic Arabidopsis thaliana

[0065] Seeds of wild-type Col, tt19 mutant, and 35S::CtGSTF3 / tt19 complementary lines #3 and #8 were sterilized and vernalized at 4 ℃ for 3 days. After sowing and culturing in nutrient soil (humus:vermiculite:perlite = 1:1:1) for 4 weeks, stress treatment was initiated. Watering was stopped in the drought treatment group; the salt treatment group was irrigated with 400 mM NaCl solution every other day; the control group was watered normally. Phenotypic images were taken on day 10 of stress treatment, and plant leaves were collected and flash-frozen in liquid nitrogen for preservation. Each experiment was conducted with three independent biological replicates.

[0066] Phenotypic results are shown in Figure 4 A: Under normal conditions, there were no significant differences in growth status among all lines. After drought stress treatment, the tt19 mutant suffered severe wilting and yellowing of leaves, exhibiting the most severe damage; the wild type suffered less damage, and the drought tolerance of the #3 and #8 complementary lines was significantly higher than that of the tt19 mutant. Under salt stress, the tt19 mutant experienced growth inhibition and leaf chlorosis; the growth status of the #3 and #8 lines was significantly better than that of the tt19 mutant and the wild-type Col. The results indicate that the tt19 mutant is sensitive to drought and salt stress, and heterologous expression of CtGSTF3 can significantly alleviate the stress-sensitive phenotype of the mutant and improve the plant's stress resistance.

[0067] Example 9: Histochemical staining of reactive oxygen species and determination of antioxidant enzyme activity after stress treatment

[0068] DAB staining: Select mature Arabidopsis leaves and completely immerse them in 0.1 mg / mL DAB solution (prepared with PBS buffer, pH 7.4) and incubate at room temperature in the dark for 24 h; then place the leaves in 95% ethanol and boil for 10-15 min to remove chlorophyll. Replace the ethanol several times until the leaves are completely decolorized for observation and photography.

[0069] NBT staining: Mature leaves were immersed in NBT staining solution (50 mg NBT dissolved in 100 mL PBS buffer, with 0.05% Tween-20 added) and kept at room temperature in the dark for 24 h; the leaves were then boiled to decolorize and observed.

[0070] Following the instructions of the kit from Nanjing Jiancheng Bioengineering Institute, the activities of catalase (CAT), peroxidase (POD), and total superoxide dismutase (SOD) in the samples were measured.

[0071] Staining results as follows Figure 4As shown in Figure B: Under normal conditions, the levels of reactive oxygen species (ROS) accumulation were low in all lines. Drought and salt stress induced a large amount of ROS production. The leaves of the tt19 mutant showed the deepest DAB and NBT staining, and ROS accumulation was significantly higher than that of the wild type and the #3 and #8 complementary lines. Enzyme activity assays showed that there were no significant differences in antioxidant enzyme activities among the lines under normal conditions. After stress treatment, the activities of SOD, CAT, and POD generally increased. The enzyme activities of the tt19 mutant were significantly lower than those of the wild type, while the antioxidant enzyme activities of the 35S::CtGSTF3 / tt19#3 and #8 complementary lines were significantly higher than those of the tt19 mutant. These results indicate that CtGSTF3 can enhance antioxidant enzyme activity, strengthen ROS scavenging capacity, and reduce oxidative damage under stress.

[0072] Example 10: Expression of anthocyanin synthesis-related genes and detection of anthocyanin content under drought and salt stress.

[0073] The experiment was conducted using the qRT-PCR system and anthocyanin extraction and determination method described in Example 3.

[0074] See results Figure 5 Under normal culture conditions, the expression levels of anthocyanin synthesis genes were low across all lines, with no significant difference in anthocyanin accumulation. Drought and salt stress significantly induced the upregulation of anthocyanin synthesis structural genes, promoting anthocyanin accumulation. Under stress conditions, the transcriptional levels of key anthocyanin synthesis genes and the total anthocyanin content in the tt19 mutant were significantly lower than those in the wild-type Col; however, the expression of anthocyanin synthesis genes and anthocyanin accumulation significantly increased in the 35S::CtGSTF3 / tt19 #3 and #8 lines. This demonstrates that CtGSTF3 can activate the anthocyanin synthesis pathway, promote anthocyanin accumulation, and participate in the drought and salt stress response process in Arabidopsis thaliana.

[0075] Example 11 Analysis of gene expression in response to drought and salt stress

[0076] The expression levels of stress response genes were determined according to the qRT-PCR detection method in Example 3.

[0077] The results are shown in Figure 6. Under normal conditions, AtCBF4, AtADH1, AtABF3, AtRD29A, AtRD29B, and AtSOS2 maintained low expression levels in all lines, with no significant differences among lines. Drought and salt stress significantly induced the upregulation of these genes. Under stress conditions, the expression levels of six stress-response genes in the tt19 mutant were significantly lower than those in the wild-type Col; while the 35S::CtGSTF3 / tt19 #3 and #8 lines significantly restored the transcription levels of these genes. The results indicate that CtGSTF3 can activate multiple stress response pathways and positively regulate the drought and salt stress tolerance of Arabidopsis thaliana.

[0078] The above description represents preferred embodiments of the present invention, but is not intended to limit the invention. Any modifications, reorganizations, or equivalent substitutions made based on the technical concept of the present invention should be included within the scope of protection of the present invention.

Claims

1. A CtGSTF3 gene, characterized in that, The full-length nucleotide sequence of the CtGSTF3 gene is as follows: 1) The nucleotide sequence shown in SEQ ID NO. 1; 2) The base sequence that is different from the nucleotide sequence shown in SEQ ID NO. 1 due to the degeneracy of the genetic codon.

2. The protein encoded by the CtGSTF3 gene as described in claim 1, characterized in that, The amino acid sequence of the protein is as follows: 1) The amino acid sequence shown in SEQ ID NO. 2; 2) An amino acid sequence with equivalent function formed by replacing, deleting and / or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO.

2.

3. A recombinant vector and a recombinant bacterial strain, characterized in that, The recombinant vector contains the CtGSTF3 gene of claim 1; the recombinant strain carries the recombinant vector.

4. The application of the CtGSTF3 gene as described in claim 1 in the breeding of transgenic plants with high anthocyanin content and in the selection of new plant varieties.

5. The application of the CtGSTF3 gene as described in claim 1 in plant color improvement genetic engineering.

6. The application according to claim 5, characterized in that, The CtGSTF3 gene is used to promote the synthesis of anthocyanins in plants, thereby deepening plant coloration.

7. The application according to claim 6, characterized in that, The plants mentioned include Arabidopsis thaliana.

8. The application of the CtGSTF3 gene according to claim 1 in improving plant resistance to abiotic stress; preferably, the abiotic stress includes drought stress and salt stress.

9. The application of the CtGSTF3 gene as described in claim 1 in plant stress resistance improvement genetic engineering.

10. The application according to claim 9, characterized in that, The CtGSTF3 gene is used to construct drought-resistant and salt-tolerant transgenic plants.

11. The application according to claim 10, characterized in that, The plants mentioned include Arabidopsis thaliana.

12. The application as described in claims 5 and 9, characterized in that, The implementation method includes the following steps: (1) constructing a recombinant plant expression vector containing the gene of claim 1; (2) introducing the recombinant plant expression vector into plant tissues or plant cells; (3) overexpressing the CtGSTF3 gene in plant cells or plant tissues.

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