Salt-tolerant gene ThGPX4 and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a salt-tolerant gene ThGPX4 and application thereof. The invention provides a salt-tolerant gene ThGPX4. The salt-tolerant gene ThGPX4 is used for coding an amino acid sequence as shown in SEQ ID NO: 2. The invention finds that the ThGPX4 gene can significantly improve the salt tolerance of arabidopsis thaliana after overexpression in arabidopsis thaliana, the salt-tolerant gene ThGPX4 is constructed into a plant expression vector, genetic transformation is performed through an agrobacterium tumefaciens-mediated genetic transformation method, and a new germplasm with a salt-tolerant characteristic can be effectively obtained. The method is of great significance to cultivation of new varieties of excellent crops and wide application in production.

Description

A salt-tolerant gene ThGPX4 and its applications Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a salt-tolerant gene ThGPX4 and its applications. Background Technology

[0002] Currently, severe soil salinization severely limits plant yield and quality, and also threatens food security and ecological balance. Traditional agronomic measures (such as freshwater leaching and soil amendment) can alleviate the problem locally, but they are costly and difficult to apply on a large scale and sustainably. Conventional breeding methods have made slow progress in cultivating highly salt-tolerant plant varieties, mainly due to the complex genetic characteristics of salt tolerance—a quantitative trait controlled by multiple genes, involving multiple synergistic physiological pathways such as ion compartmentalization, synthesis of osmotic regulators, and activation of antioxidant systems. Therefore, conventional breeding methods are generally ineffective in breeding salt-tolerant crops.

[0003] Currently, gene breeding technology, due to its advantages of precision, efficiency, and controllability, has overcome the limitations of traditional breeding methods that rely on random hybridization and natural mutation. Therefore, gene breeding technology has become a hot topic in salt-tolerant crop breeding. However, in the current field of gene breeding technology, salt-tolerant gene resources are still lacking. Therefore, how to discover key salt-tolerant genes has become an urgent problem to be solved in this field.

[0004] Metasequoia (Taxodium hybrid zhongshanshan) is a plant belonging to the genus Taxodium Rich., which possesses advantages such as salt and alkali tolerance, rapid growth, waterlogging tolerance, disease and pest resistance, and excellent wood quality. Therefore, how to study the salt tolerance mechanism of Metasequoia, discover key salt tolerance genes, and enhance the plant's salt tolerance through genetic engineering technology has become an urgent problem to be solved in this field. Summary of the Invention

[0005] To address the lack of effective genes for regulating plant salt tolerance in existing technologies, this invention provides a salt tolerance gene ThGPX4 and its application, specifically including the following technical solution: This invention provides a salt tolerance gene ThGPX4, which encodes the amino acid sequence shown in SEQ ID NO:2.

[0006] Preferably, the nucleotide sequence of the salt tolerance gene ThGPX4 is shown in SEQ ID NO:1.

[0007] The present invention also provides biological materials for overexpressing the salt-tolerant gene ThGPX4 as described above, the biological materials comprising primer pairs for amplifying the salt-tolerant gene ThGPX4 and transformants containing the salt-tolerant gene ThGPX4.

[0008] Preferably, the primer pair comprises sequences as shown in SEQ ID NO:3 and SEQ ID NO:4.

[0009] Preferably, the transformant includes any one or more of the following: a recombinant vector overexpressing the salt tolerance gene ThGPX4, a recombinant microorganism overexpressing the salt tolerance gene ThGPX4, a transgenic cell line overexpressing the salt tolerance gene ThGPX4, and a transgenic plant tissue overexpressing the salt tolerance gene ThGPX4.

[0010] Preferably, the recombinant vector includes a backbone vector, the backbone vector including pCAMBIA1301; the recombinant microorganism includes a basic microorganism, the basic microorganism including Agrobacterium.

[0011] This invention also provides the application of the salt-tolerant gene ThGPX4 or biomaterials as described above in improving plant salt tolerance and / or in salt-tolerant plant breeding.

[0012] Preferably, the improvement of plant salt tolerance includes positively regulating the salt tolerance gene ThGPX4 through transgenic means to improve plant salt tolerance.

[0013] Preferably, the salt-tolerant plant breeding includes positively regulating the salt-tolerant gene ThGPX4 in plants to obtain plant varieties with improved salt tolerance.

[0014] Preferably, the plant includes Arabidopsis thaliana and / or Metasequoia glyptostroboides.

[0015] The beneficial effects of this invention are as follows: This invention provides a salt-tolerant gene, ThGPX4, which encodes the amino acid sequence shown in SEQ ID NO:2. Verification through examples shows that overexpression of this gene in plants significantly improves their salt tolerance. Constructing the ThGPX4 gene into a plant expression vector and performing genetic transformation using Agrobacterium-mediated transformation effectively yields new germplasm with salt-tolerant characteristics. This invention is of great significance for the breeding of superior new crop varieties and their widespread application in production. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 shows the pCAMBIA1301 plasmid vector map; Figure 2 shows the subcellular localization of the ThGPX4 gene in Arabidopsis protoplasts; where GFP (Green Fluorescent Protein) is the empty green fluorescence channel; Auto (Chlorophyll Autofluorescence Channel) is the empty chloroplast autofluorescence channel; Bright (Bright field) is the empty bright field; Merged (Merge field) is the superposition of the empty bright field and fluorescence channel; Figure 3 shows the relative expression level of the ThGPX4 gene in different tissues; where a is a bar chart of the relative expression level of the ThGPX4 gene in different tissues; b is the callus tissue of Metasequoia glyptostroboides; c is the dominant embryo of Metasequoia glyptostroboides; d is the cotyledonary embryo of Metasequoia glyptostroboides; e is the stem segment of Metasequoia glyptostroboides; f is the leaf of Metasequoia glyptostroboides; Figure 4 shows the phenotypic observation of ThGPX4 overexpressing Arabidopsis thaliana; where A is the soil culture condition without salt stress treatment, 28 A) Comparison of leaf size between wild-type plants (WT) and ThGPX4-overexpressing plants (OE-1, OE-2, and OE-3) at 28 days; B) Bar chart of single-plant fresh weight of wild-type Arabidopsis and ThGPX4-overexpressing Arabidopsis lines at 28 days; C) Bar chart of single-plant fresh weight of wild-type Arabidopsis and ThGPX4-overexpressing Arabidopsis at 28 days. Figure 5 shows the phenotypic comparison between ThGPX4-overexpressing Arabidopsis and wild-type Arabidopsis under salt stress treatment; Figure 6 shows the root growth of ThGPX4-overexpressing Arabidopsis and wild-type Arabidopsis under salt stress treatment; where A is the comparison of roots without salt stress treatment; B is the comparison of root length under salt stress treatment; C is the bar chart of the maximum root length of Arabidopsis in different groups before and after salt stress; D is the bar chart of the number of lateral roots of Arabidopsis in different groups before and after salt stress; Figure 7 shows the determination of physiological activities of Arabidopsis under salt stress treatment; where A is the bar chart of SOD activity of wild-type and ThGPX4-overexpressing Arabidopsis after 10 days of salt stress treatment; B is the bar chart of GSH content of wild-type and ThGPX4-overexpressing Arabidopsis after 10 days of salt stress treatment; C is the bar chart of the maximum root length of Arabidopsis in different groups before and after salt stress treatment; D is the bar chart of the number of lateral roots of Arabidopsis in different groups before and after salt stress treatment; Figure 7 shows the determination of physiological activities of Arabidopsis under salt stress treatment; where A is the bar chart of SOD activity of wild-type and ThGPX4-overexpressing Arabidopsis after 10 days of salt stress treatment; B is the bar chart of GSH content of wild-type and ThGPX4-overexpressing Arabidopsis after 10 days of salt stress treatment; C is the bar chart of the maximum root length of Arabidopsis in different groups before and after salt stress treatment; D is the bar chart of the maximum root length of Arabidopsis after 10 days of salt stress treatment; Figure 8 shows the bar chart of CAT activity in wild-type and ThGPX4-overexpressing Arabidopsis thaliana after d; Figure 8 shows the GUS staining and physiological index determination of ThGPX4 after transient transformation in embryogenic callus.

[0018] In the figures, A shows the staining of uninfected embryogenic callus under a stereomicroscope after GUS staining; B shows the staining of callus infected for 48 h under a stereomicroscope after GUS staining; C shows the CAT activity of CK and ThGPX4 transiently expressed callus after 48 h of salt stress treatment; D shows the SOD activity of CK and ThGPX4 transiently expressed callus after 48 h of salt stress treatment; E shows the MDA content of CK and GPX4 transiently expressed callus after 48 h of salt stress treatment; and F shows the T-AOC activity of CK and GPX4 transiently expressed callus after 48 h of salt stress treatment. Detailed Implementation

[0019] The present invention provides a salt tolerance gene ThGPX4, which encodes the amino acid sequence shown in SEQ ID NO:2.

[0020] As one embodiment, the nucleotide sequence of the salt tolerance gene ThGPX4 described in this invention is shown in SEQ ID NO:1.

[0021] The present invention also provides biological materials for overexpressing the salt-tolerant gene ThGPX4 as described above, the biological materials comprising primer pairs for amplifying the salt-tolerant gene ThGPX4 and transformants containing the salt-tolerant gene ThGPX4.

[0022] In one embodiment, the primer pair includes sequences as shown in SEQ ID NO:3 and SEQ ID NO:4. In one embodiment, the transformant includes any one or more of the following: a recombinant vector overexpressing the salt tolerance gene ThGPX4, a recombinant microorganism overexpressing the salt tolerance gene ThGPX4, a transgenic cell line overexpressing the salt tolerance gene ThGPX4, and a transgenic plant tissue overexpressing the salt tolerance gene ThGPX4. In one embodiment, the transgenic cell line overexpressing the salt tolerance gene ThGPX4 and the transgenic plant tissue overexpressing the salt tolerance gene ThGPX4 are non-stable genetic cell lines and plant tissues. In one embodiment, the recombinant vector includes a backbone vector. In one embodiment, the backbone vector includes a plasmid vector. In one embodiment, the plasmid vector includes pCAMBIA1301. In one embodiment, the recombinant microorganism includes a basic microorganism. In one embodiment, the basic microorganism includes bacteria. In one embodiment, the bacteria includes Agrobacterium.

[0023] This invention also provides the application of the salt-tolerant gene ThGPX4 or biomaterials as described above in improving plant salt tolerance and / or in salt-tolerant plant breeding.

[0024] In one embodiment, improving plant salt tolerance includes positively regulating the salt tolerance gene ThGPX4 through transgenic methods to enhance plant salt tolerance. In another embodiment, salt-tolerant plant breeding includes positively regulating the salt tolerance gene ThGPX4 in plants to obtain plant varieties with improved salt tolerance. In one embodiment, the positive regulation includes overexpression. In one embodiment, the plant includes Arabidopsis thaliana and / or Metasequoia glyptostroboides.

[0025] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a salt-tolerant gene ThGPX4 and its applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1: Sequence Information of the *Taxus chinensis* ThGPX4 Gene. The full-length open reading frame (OPF) sequence of the *Taxus chinensis* ThGPX4 gene in this invention is 762 bp, as shown in SEQ ID NO:1. Based on the ORF sequence, the amino acid sequence of the *Taxus chinensis* transcription factor ThGPX4 protein was deduced, consisting of 253 amino acids, as shown in SEQ ID NO:2. The molecular weight is 28273.36 KD, the isoelectric point (pI) is 8.09, and it possesses a most conserved Thioredoxin-like domain.

[0027] SEQ ID NO:1:5'--3'。

[0028] SEQ ID NO:2:MLPPTVTWRCFTLPHFINIAKALCIQYKPINKFSKPHNFHTIAYCNINPPTGCLPIDSTVPTKRNHPLPIGFSLFSSTNFSTMDGSSSEGHANVHDFTVKDIRGNDVDLSIYKGKALLIVNVA SECGLTTSNYKEMNELYAKYKDQGLEILAFPCNQFAGEEPGDNEQIAEVACTRFKAEFPVFEKVEVNGDNTAPIYKFLKSRKGGFLGDNIKWNFTKFLVDKDGNVIDRYAPTTSPLNIEKHIKKLLGTV.

[0029] Example 2 Cloning of the ThGPX4 gene of Metasequoia glyptostroboides 1. Extraction of target fragment RNA The material used for extraction was Metasequoia glyptostroboides 406 leaves. After collecting Metasequoia glyptostroboides 406 leaves, they were immediately placed in liquid nitrogen for storage and later use.

[0030] Total RNA was extracted from leaves using the Prometheus RNA Extraction Kit (LS1040), with specific procedures outlined in the kit's instruction manual. After extraction, the purity and concentration of total RNA were assessed, and RNA bands were analyzed using agarose gel electrophoresis. The sample RNA was then reverse transcribed into cDNA (Novozymes, R312-01), with specific procedures outlined in the instruction manual. Specific primers for the full-length target gene sequence were designed using NCBI, and the primer sequences are shown in Table 1.

[0031] Table 1. Primer sequences for PCR amplification

[0032] The target fragment was obtained using the Phanta Max Super-Fidelity DNA Polymerase kit. The PCR reaction system and amplification system are shown in Tables 2-3.

[0033] Table 2 PCR reaction system

[0034] Table 3 PCR amplification system

[0035] After the PCR reaction was completed, the size of the target band was detected by 1% agarose gel electrophoresis, and the target band was excised and recovered. The specific recovery steps were performed according to the Qingke DNA Gel Recovery Kit.

[0036] 2. Ligation and transformation of the target fragment (1) The cloning vector used for ligation was PMD19-T Vector, which was purchased from TaKara. The reaction system is shown in Table 4.

[0037] Table 4. Reaction systems for the target fragment and cloning vector.

[0038] Gently pipette and mix well, centrifuge at low speed, and incubate overnight at 16°C.

[0039] (2) The transformation of the ligation product was carried out using DH5α Escherichia coli competent cells (TIANGEN). The specific operation steps are shown in the instruction manual.

[0040] (3) Pick a single colony and place it in LB medium containing 50 mg / L ampicillin. Incubate at 37°C and 220 rpm. Perform preliminary identification of the bacterial culture using M13 universal primers. The primer sequences are shown below. Then, send the preliminarily identified positive clones to Qingke Biotechnology for sequencing.

[0041] F (SEQ ID NO:5): 5'-CAGGGTTTTCCCAGTCACG-3'; R (SEQ ID NO:6): 5'-GAGCGGATAACAATTTCACAC-3'; (4) After successful sequencing, plasmids were extracted according to the kit instructions (Vazyme product number DC211-02). PCR reaction, gel electrophoresis, and gel extraction were then performed to recover the target band.

[0042] (5) The plasmid vector pCAMBIA1301 used in this experiment is shown in Figure 1. The restriction sites are NcoI and BglII, and the restriction system is shown in Table 5: Table 5 Restriction System

[0043] The above enzyme digestion system was reacted at 37°C for 3 hours, and then at 65°C for 30 minutes.

[0044] (6) After enzyme digestion, single-fragment recombination was performed using the ClonExpress II One StepCloning Kit. Before preparing the recombination reaction system, the linearized vector and the inserted fragment needed to be diluted. The amount of each component added should not be less than 1 μL. The reaction system is shown in Table 6.

[0045] Table 6 Reorganization System

[0046] The above system was reacted at 37°C for 30 min, and then placed at 4°C.

[0047] (7) Transformation of the ligation product was performed using DH5α Escherichia coli competent cells (TIANGEN). The specific operating steps are described in the instruction manual. Single colonies were picked and placed in LB medium containing kanamycin. The colonies were cultured at 28°C and 200 rpm. After preliminary identification by PCR, the colonies were sent to Qingke Biotechnology for sequencing.

[0048] (8) After successful sequencing, plasmids were extracted and transformed into Agrobacterium competent cells. GV3101 (Kulaibo) was selected as the Agrobacterium competent cells, and the transformation steps were performed according to the instructions. Single colonies were picked and placed in LB medium containing 50 mg / L kanamycin and cultured at 28℃ and 200 rpm. The bacterial culture was initially identified by PCR, and then the positive clones were sent to Qingke Biotechnology for sequencing.

[0049] (9) The successfully sequenced plasmid was named the 35S-GPX4-GFP vector and used for subsequent genetic transformation.

[0050] Example 3: Subcellular Localization Analysis of the ThGPX4 Gene in Arabidopsis Protoplasts To further understand the location of the ThGPX4 protein in vivo, the 35S-GPX4-GFP vector constructed in Example 2 was transiently transformed into Arabidopsis protoplasts to observe and locate the subcellular distribution of this protein. The specific procedures were as follows: Leaves from Arabidopsis seedlings grown in soil for 3-4 weeks were vacuum-permeable with 10 mL of enzymatic hydrolysate and sealed. The leaves were placed under low light conditions at 23℃ for 3 h for enzymatic hydrolysis. After hydrolysis, the mixture was filtered and centrifuged at 400 rpm for 5 min to collect the precipitate. The precipitate was washed twice with W5 solution, then centrifuged after an ice bath for 30 min, the supernatant was discarded, and the precipitate was resuspended in MMG solution to a microscopic density of 20-40 cells / field at 40x magnification.

[0051] The raw material content of W5 solution is as follows: 154 mM NaCl, 125 mM CaCl2, 5 mM KCl and 5 mM MES (pH 5.7).

[0052] The MMG solution system consisted of: 10 mM MgCl2, 5 mM MES, and 0.4 M mannitol (pH 5.7).

[0053] For standard localization experiments, 10 μL of the target gene plasmid (35S-GPX4-GFP vector) + 100 μL of protoplasts + 110 μL of 40% PEG4000 solution were used. After mixing, the mixture was incubated at 22.5℃ for 15-20 min, the reaction was terminated by W5 and the sample was washed twice. Finally, the sample was resuspended in W5 and cultured at 23℃ under low light for 12-16 h.

[0054] Cell pellets were collected by centrifugation at 400 rpm for 5 min before observation, and images were acquired and analyzed using a laser confocal microscope. GFP was used as a positive control, and the results are shown in Figure 2. It can be seen that, based on the green fluorescence signal of the target gene, the ThGPX4 gene signal is widely expressed in both the nucleus and cytoplasm.

[0055] Example 4: Expression of the ThGPX4 gene in different tissues of *Taxus chinensis* 406 was investigated. *Taxus chinensis* 406 callus, dominant embryo, cotyledonary embryo, stem segment, and leaves were used as sample materials. These sample materials were flash-frozen in liquid nitrogen and stored at -80℃. RNA extraction, determination of RNA purity and concentration, and acquisition of cDNA were performed as described in Example 2. Specific primers were designed for real-time quantitative PCR analysis of gene expression levels in different tissues, using APRT. a The gene used was the internal reference gene (accession number of the internal reference gene in the NCBI database: KX431853, published in the literature "Wang Z, et al. Identification of suitable reference genes in Taxodium 'Zhongshanshan' underabiotic stresses Trees.. 2017 Oct;31(5):1519-1530. DOI: 10.1007 / s00468-017-1566-y.), and the primers are shown in Table 7. The results are shown in Figure 3. It can be seen that the expression level of the ThGPX4 gene is higher in the dominant embryo and stem segment, and the expression level is lowest in the cotyledon embryo.

[0056] Table 7 Specific Primers

[0057] In Example 5, the transformation of Arabidopsis thaliana with the ThGPX4 gene from Taxodium alpinum involved the inflorescence infection method. Specifically, before infection, all open flowers in the Arabidopsis were removed, and the Agrobacterium tumefaciens strain prepared in Example 2, capable of overexpressing the ThGPX4 gene, was cultured to OD200. 600The bacterial culture was centrifuged at 4°C and 5000 rpm for 10 min until the bacterial mass reached approximately 0.8. The bacterial culture was then resuspended in MS liquid medium containing 20 g / L sucrose, and Silwet-77 was added. The mixture was shaken well to obtain the infection solution. Arabidopsis inflorescences were immersed in the infection solution for approximately 30 s, bagged, and cultured in the dark for 24 h. Afterward, they were placed under normal light conditions and cultured for another 3-4 weeks to harvest T0 generation seeds. T0 generation seeds were germinated on a selection medium supplemented with 50 mg / L hygromycin and further cultured in soil to harvest T1 generation seeds. Culture continued until T3 generation seeds were harvested. T3 generation seeds were cultured for 28 days from sowing, and phenotypic observations were performed, as shown in Figure 4A. On day 28 of growth, the fresh weight of individual Arabidopsis plants was measured, as shown in Figure 4B. On day 28 of growth, the leaf area of ​​individual Arabidopsis plants was measured, as shown in Figure 4C.

[0058] As shown in Figure 4, there are differences in leaf size between wild-type Arabidopsis and Arabidopsis plants overexpressing the ThGPX4 gene. After repeated comparisons of multiple lines, it was found that the leaf area of ​​the ThGPX4 gene overexpressing plants was significantly larger than that of the wild-type plants (P < 0.05), and the fresh weight of the overexpressing lines was greater than that of the wild type.

[0059] Example 6: Phenotypic Analysis of Arabidopsis thaliana Overexpressing the ThGPX4 Gene. Seeds from three T3 generation transgenic lines (OE-1, OE-2, OE-3) obtained in Example 5, homozygous ThGPX4 gene overexpressing lines, and wild-type seeds (WT) were taken. 1 ml of water was added, and the seeds were vernalized at 4°C for 72 hours. The seeds were then sown in sterilized nutrient soil and placed in an incubator at 20-23°C with 16 hours of light / 8 hours of darkness until germination. Once the Arabidopsis reached a robust two-leaf stage, they were transplanted. After 21 days, the seedlings were subjected to salt stress treatment by applying 4 ml of 50 mmol / L NaCl solution at the same time each day. The Arabidopsis were observed on days 1, 5, 10, and 15 of the salt stress treatment, and phenotypic differences were recorded. The results are shown in Figure 5.

[0060] As shown in Figure 5, leaf yellowing began to appear 5 days after salt stress treatment. By day 10, a clear difference between the wild-type and overexpression lines was observed. At this point, the growth of wild-type Arabidopsis was significantly inhibited, and its leaves showed significant yellowing, while the yellowing of leaves in different overexpression lines was not significant. After day 15, growth was inhibited in both wild-type and overexpression lines. However, compared with the overexpression plants, the wild-type plants showed significant leaf yellowing and stopped growing, while the overexpression plants were less affected by salt stress and grew well. Therefore, it can be seen that overexpression of the ThGPX4 gene significantly improves the salt stress tolerance of Arabidopsis.

[0061] Seeds of the three transgenic lines (OE-1, OE-2, and OE-3) and wild-type seeds were surface-sterilized with 0.4% sodium hypochlorite and sown in 1 / 2 MS medium. After cold treatment at 4°C for 2 days, the seedlings were transferred to an incubator. After 7 days, the seedlings were transferred to 1 / 2 MS medium containing 50 mmol / L NaCl. Phenotypic differences were observed and recorded after 14 days, and the results are shown in Figure 6.

[0062] As shown in Figure 6, growth was inhibited in both the wild-type and ThGPX4-overexpressing lines under 50 mmol / L NaCl stress. However, compared to the wild-type, the root length of ThGPX4-overexpressing Arabidopsis thaliana was superior, demonstrating stronger salt tolerance. Under 50 mmol / L NaCl stress, the average maximum root lengths of OE-1, OE-2, and OE-3 were 4.38 cm, 4.47 cm, and 4.57 cm, respectively, which were greater than the 3.73 cm of WT. In terms of lateral roots, OE-1, OE-2, and OE-3 had more lateral roots than WT, demonstrating adaptation to salt stress and thus enabling the plants to obtain more nutrients to maintain growth. Therefore, growth indicators show that ThGPX4-overexpressing Arabidopsis thaliana exhibits superior salt tolerance.

[0063] Example 7 Physiological analysis of Arabidopsis thaliana overexpressing the ThGPX4 gene: The physiological indicators (SOD, GSH and CAT enzyme activities) of Arabidopsis thaliana plants after 10 days of salt stress treatment in Example 6 were measured according to conventional detection methods. The results are shown in Figures A to C in Figure 7.

[0064] Superoxide dismutase (SOD) activity in plants is a core indicator for measuring the ability of plant cells to scavenge harmful free radicals, their antioxidant capacity, and overall stress resistance. As shown in Figure 7, the SOD activity of wild-type Arabidopsis thaliana was significantly lower than that of overexpressing plants, indicating that overexpression of the ThGPX4 gene can enhance the antioxidant capacity and the ability to scavenge harmful free radicals in plants.

[0065] GSH content represents the comprehensive ability of plant cells to resist oxidative damage, detoxify harmful substances, and regulate growth and development. It is a key indicator for measuring plant health and stress resistance. As shown in Figure 7, the GSH content in overexpressing plants was higher than that in wild-type plants. This indicates that overexpression of the ThGPX4 gene can enhance the plant's comprehensive ability to resist damage, detoxify, and grow.

[0066] CAT activity represents the plant's ability to scavenge toxic substances such as hydrogen peroxide (H2O2) and is a key indicator for measuring the plant's antioxidant and detoxification system. As shown in Figure 7, the CAT activity in the wild type was also lower than that in the overexpressing plant. This indicates that overexpression of the ThGPX4 gene can enhance the plant's antioxidant and detoxification capabilities.

[0067] Example 8: Transient transformation and physiological index determination of ThGPX4 in embryogenic callus. Transient transformation includes steps such as Agrobacterium activation, infection, co-culture, and destering.

[0068] 8.1 Agrobacterium activation: Glycerol-containing bacteria with empty vector and ThGPX4 gene, stored at -80℃, were removed and thawed on ice. A small amount of glycerol-containing bacterial suspension was streaked onto LB solid medium containing 50 mg / L kanamycin using an inoculation ring. The ring was then inverted and incubated in the dark at 28℃ for 48 h. Once a single colony appeared, it was picked up with a sterile pipette tip and placed in LB liquid medium containing 50 mg / L kanamycin. The culture was then incubated at 28℃ with shaking at 200 r / min for 18 h. After the bacterial suspension became turbid, 200 μL of the bacterial suspension was transferred to 100 ml of LB liquid medium containing 50 mg / L kanamycin. The culture was then incubated at 28℃ with shaking at 200 r / min for 16 h. The OD value was measured every 2 h during the incubation period. 600 When the value reaches 0.8, stop the culture.

[0069] 8.2 Infection and co-culture (1) Raw materials and contents of conversion solution: 1 / 2MS, 270 mM / L mannitol, 0.5 mg / L KT, 0.5 mg / L NAA, 120 μM / L AS, 20 μM / L 5-azaC, 200 mg / L DTT, 0.02% Tween, 40 mM / L calcium chloride and 20 g / L sucrose.

[0070] (2) Raw materials and content of hypertonic treatment solution: 1 / 2MS medium containing 25% sucrose, pH 5.8.

[0071] (3) Raw materials and contents of sterilization solution: 120 μM / L AS, 0.5 mg / L NAA, 2 mg / L 6-BA, 200 mg / L LTT, 40 mM / L calcium chloride and 30 g / L sucrose.

[0072] (4) Raw materials and contents of co-culture medium: MS, 150 μM / L AS, 200 mg / L DTT, 10 g / L sucrose and 2 g / L gel.

[0073] Operating steps: OD 600 Transfer the bacterial culture with an OD value of 0.8 to a sterile centrifuge tube and centrifuge at 4°C for 15 min. Discard the supernatant and retain the turbid bacterial culture precipitate. Add a small amount of conversion medium to the centrifuge tube containing the bacterial culture precipitate and vortex to completely dissolve the precipitate. Continue to add conversion medium in small amounts several times until the OD value reaches 0.8. 600The value reached 0.7. The callus was completely immersed in the hypertonic treatment solution for 15 minutes, then transferred to an Erlenmeyer flask for later use. Two-thirds of the volume of OD was added to the Erlenmeyer flask. 600 The bacterial transformation medium was prepared at 0.7 g / L and cultured at 28°C for 1 h at 90 rpm. The remaining 1 / 3 volume of the bacterial transformation medium was then added, and the culture was continued at 90 rpm for 0.5 h. After the culture, the bacterial transformation medium containing callus tissue was filtered out using a 50-mesh sterile cell sieve. The callus tissue was then transferred back to an Erlenmeyer flask and quickly rinsed with sterilization solution for 1 min. The surface moisture was blotted dry with sterile filter paper, and the tissue was inoculated onto a co-culture medium containing 50 mmol / L NaCl and cultured in the dark for 48 h.

[0074] 8.3 GUS staining: Callus tissue that had been cultured in the dark for 48 h was placed in a test tube containing GUS staining solution. The test tube was wrapped with aluminum foil and placed in the dark. After staining at room temperature for 18 h, the staining of the callus tissue was observed using a stereomicroscope and a microscope. The results are shown in Figures A and B in Figure 8.

[0075] 8.4 Physiological Analysis of ThGPX4 Overexpression Callus Transiently transformed callus tissues were collected, and the enzyme activities of CAT, SOD, MDA, and T-AOC in both unexpressed (CK) and ThGPX4 overexpressed callus tissues were measured. The results are shown in Figures 8C-F. It can be seen that after transient ThGPX4 expression, CAT and SOD were significantly higher than in the control group, demonstrating a significant improvement in antioxidant capacity and the ability to scavenge toxic substances such as hydrogen peroxide (H2O2). MDA was downregulated, indicating a reduction in the accumulation and damage of reactive oxygen species. Finally, T-AOC was significantly upregulated, indicating an improvement in the total antioxidant capacity of the tissue, verifying the significant enhancement of salt tolerance after transient expression of ThGPX4 in *Taxus chinensis*.

[0076] In summary, the above results confirm that the ThGPX4 gene can enhance the salt tolerance of plants, and therefore can be used to improve the plant's adaptability to salt stress, making it suitable for salt-stress-resistant breeding. In conclusion, this invention provides a ThGPX4 gene from *Taxus chinensis*. Verification through examples shows that overexpression of this gene in *Arabidopsis thaliana* significantly improves its salt tolerance. Constructing the salt-tolerant gene ThGPX4 into a plant expression vector and performing genetic transformation using Agrobacterium-mediated transformation can effectively obtain new germplasm with salt-tolerant characteristics. This invention is of great significance for the breeding of superior new crop varieties and their widespread application in production.

[0077] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments without creative effort, as shown in these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A salt tolerance gene ThGPX4, characterized in that, The salt tolerance gene ThGPX4 encodes the amino acid sequence shown in SEQ ID NO:

2.

2. The salt tolerance gene ThGPX4 as described in claim 1, characterized in that, The nucleotide sequence of the salt tolerance gene ThGPX4 is shown in SEQ ID NO:

1.

3. A biomaterial for overexpressing the salt tolerance gene ThGPX4 as described in claim 1, characterized in that, The biological material includes primer pairs for amplifying the salt tolerance gene ThGPX4 and transformants containing the salt tolerance gene ThGPX4.

4. The biomaterial as described in claim 3, characterized in that, The primer pairs include sequences as shown in SEQ ID NO:3 and SEQ ID NO:

4.

5. The biomaterial as described in claim 3, characterized in that, The transformant includes any one or more of the following: a recombinant vector overexpressing the salt tolerance gene ThGPX4, a recombinant microorganism overexpressing the salt tolerance gene ThGPX4, a transgenic cell line overexpressing the salt tolerance gene ThGPX4, and a transgenic plant tissue overexpressing the salt tolerance gene ThGPX4.

6. The biomaterial as described in claim 3, characterized in that, The recombinant vector includes a backbone vector, which includes pCAMBIA1301; the recombinant microorganism includes a basic microorganism, which includes Agrobacterium.

7. The application of the salt-tolerant gene ThGPX4 as described in claim 1 or 2, or the biomaterial as described in any one of claims 3 to 6, in improving plant salt tolerance and / or in the breeding of salt-tolerant plants.

8. The application as described in claim 7, characterized in that, The improvement of plant salt tolerance includes positively regulating the salt tolerance gene ThGPX4 through transgenic methods to enhance plant salt tolerance.

9. The application as described in claim 7, characterized in that, The salt-tolerant plant breeding includes positively regulating the salt-tolerant gene ThGPX4 in plants to obtain plant varieties with improved salt tolerance.

10. The application as described in any one of claims 7 to 9, characterized in that, The plants mentioned include Arabidopsis thaliana and / or Metasequoia glyptostroboides.

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