Application and method of SlGRXC9 gene in improvement of cadmium stress tolerance of tomato

Abstract

The invention relates to the technical field of plant genetic engineering and agricultural planting, in particular to application and method of an SlGRXC9 gene in improvement of tomato cadmium stress tolerance, the number of the SlGRXC9 gene is Solyc07g053550, the core coding region sequence of the SlGRXC9 gene is SEQ ID NO.1, and the coded protein sequence is SEQ ID NO.2; through construction of SlGRXC9 overexpression transgenic tomatoes, heterologous expression of protein encoded by the gene in plant cells or use of the gene as a molecular breeding target, the system activity of antioxidant enzymes such as SOD, POD, CAT and GR in tomatoes can be enhanced, cadmium stress induced H2O2 and O2-accumulation and membrane lipid peroxidation damage are reduced, and absorption and accumulation of the tomatoes to cadmium ions are not affected; according to the method, the growth ability and physiological stability of the tomatoes under cadmium stress are remarkably improved, the risk of nutrient imbalance is avoided, the hereditary stability is high, and the method can be widely applied to safe production of the tomatoes in cadmium-contaminated soil and creation of stress-tolerant germplasm.

Description

Application and Method of SlGRXC9 Gene in Improving Tomato Cadmium Stress Tolerance Technical Field

[0001] This invention relates to the fields of plant genetic engineering and agricultural planting technology, specifically to the application and method of the SlGRXC9 gene in improving the tolerance of tomatoes to cadmium stress. Background Technology

[0002] Cadmium (Cd) is a highly toxic heavy metal pollutant to both plants and human health, widely present in farmland soils affected by industrial emissions and fertilizer use. Tomatoes, as an important greenhouse vegetable crop, are susceptible to cadmium pollution stress during production, leading to inhibited plant growth, reduced photosynthesis, and excessive accumulation of reactive oxygen species (ROS). In severe cases, it can cause cell membrane lipid peroxidation and even plant death. Current research and applications to improve plant cadmium tolerance mainly focus on three technical pathways: reducing cadmium absorption or translocation to aboveground parts by regulating metal transport proteins; isolating cadmium in vacuoles using chelating proteins or metallothioneins; and alleviating cadmium-induced oxidative stress through exogenous application of antioxidants. These technical pathways have been widely explored and applied in related research and practice.

[0003] The aforementioned existing technical approaches generally have significant shortcomings. For example, the regulation of cadmium transport or absorption often affects the plant's absorption of other essential micronutrients, potentially leading to nutrient imbalances. The complex mechanisms of metal chelation and isolation result in insufficient stability for genetic improvement based on these mechanisms, making it difficult to meet practical application needs. Exogenous antioxidant treatments are costly and lack sustainability, making them unsuitable for large-scale agricultural production. Therefore, there is an urgent need to discover a key regulatory factor that does not affect cadmium absorption and transport but significantly enhances the plant's own antioxidant capacity, thereby improving cadmium tolerance, to meet the requirements of safe, efficient, and heritably usable agricultural applications. Summary of the Invention

[0004] The purpose of this invention is to provide an application and method of the SlGRXC9 gene in improving the tolerance of tomatoes to cadmium stress, so as to solve the problems mentioned in the background art.

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

[0006] An SlGRXC9 gene, the core coding region sequence of which is SEQ ID NO.1, and the gene number is Solyc07g053550.

[0007] As a further aspect of the present invention: the protein sequence encoded by the gene is SEQ ID NO.2.

[0008] A method for improving tomato tolerance to cadmium stress using the SlGRXC9 gene involves enhancing the expression or activity of the SlGRXC9 gene in tomatoes, thereby increasing the activity of the tomato's endogenous antioxidant enzyme system, reducing cadmium stress-induced reactive oxygen species accumulation and membrane lipid peroxidation damage, and thus improving tomato's tolerance to cadmium stress.

[0009] As a further aspect of the present invention: the method of enhancing SlGRXC9 gene expression is to construct an SlGRXC9 gene overexpression vector, introduce the overexpression vector into tomato cells or tissues, and obtain transgenic tomatoes that overexpress the SlGRXC9 gene.

[0010] As a further aspect of the present invention, the overexpression vector is introduced into tomato cells or tissues using an Agrobacterium-mediated transformation method.

[0011] As a further aspect of the present invention: the endogenous antioxidant enzyme system includes superoxide dismutase, peroxidase, catalase and glutathione reductase.

[0012] As a further aspect of the present invention, the method does not affect the absorption and accumulation of cadmium ions by tomatoes.

[0013] An application of the SlGRXC9 gene described above in improving the tolerance of tomatoes to cadmium stress, wherein the SlGRXC9 gene is used for the safe production of tomatoes in cadmium-contaminated soil or for the creation of cadmium-tolerant tomato germplasm.

[0014] As a further aspect of the present invention, the application method is to use the SlGRXC9 gene as a molecular breeding target to screen or cultivate tomato varieties resistant to cadmium stress.

[0015] As a further aspect of the present invention, the application method is to plant genetically modified tomatoes obtained by the method described in claim 5.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] It significantly improves the growth capacity and physiological stability of tomatoes under cadmium stress; it does not change the cadmium ion content in the plant, avoiding the risk of nutrient imbalance caused by changes in transport; it works by enhancing the endogenous antioxidant system, has high genetic stability, and is suitable for long-term breeding use; it can be widely applied to the safe production of tomatoes and the creation of stress-resistant germplasm under cadmium-contaminated soil conditions. Attached Figure Description

[0018] Figure 1 shows the expression characteristics of the SlGRXC9 gene and the verification of cadmium resistance function by yeast heterologous expression in the embodiments of the present invention.

[0019] In this diagram, A shows the relative expression level of SlGRXC9 in tomato roots under 50 μM CdCl2 stress (treatment time 0-5 days); B shows the specific expression level distribution of SlGRXC9 in tomato young leaves, green-ripe fruits, color-changing fruits, and red-ripe fruits; C shows the growth curve (OD600 value changes over time) of the yeast cadmium-sensitive mutant Δycf1 (transformed into empty vector EV and SlGRXC9 gene, respectively) in media containing 15 μM, 25 μM, and 50 μM CdCl2; D shows the survival rate of the yeast mutant Δycf1 (transformed into empty vector EV and SlGRXC9 gene, respectively) in media containing different concentrations of CdCl2; and E shows the intracellular cadmium content detection results of the yeast mutant Δycf1 (transformed into empty vector EV and SlGRXC9 gene, respectively).

[0020] Figure 2 shows the construction of the SlGRXC9 transgenic tomato line and the detection of growth phenotype and photosynthetic pigment content under cadmium stress in the embodiment of the present invention.

[0021] In this diagram, A shows the relative expression level of SlGRXC9 in SlGRXC9 overexpressing transgenic tomato lines (including lines such as OE5); B shows the sequencing verification map of the SlGRXC9 CRISPR / Cas9 knockout lines; C shows a schematic diagram of the base insertion mutation in the SlGRXC9 homozygous knockout line (slgrxc9-1); D shows a comparison of leaf morphology between wild-type WT, overexpressing line OE5, and knockout line slgrxc9-1 under normal conditions; E shows the aboveground dry weight of tomatoes under 50 μM CdCl2 stress (comparing wild-type WT, overexpressing line OE5, and knockout line slgrxc9-1); F shows the underground dry weight of tomatoes under 50 μM CdCl2 stress (comparing WT, OE5, and slgrxc9-1); G shows the 50 μM CdCl2 stress. Figure 1 shows the results of chlorophyll a content detection in tomatoes under CdCl2 stress (compared to WT, OE5, and slgrxc9-1); Figure 2 shows the results of chlorophyll b content detection in tomatoes under 50 μM CdCl2 stress (compared to WT, OE5, and slgrxc9-1).

[0022] Figure 3 shows the detection effect of SlGRXC9 on reactive oxygen accumulation and membrane lipid peroxidation damage in tomatoes under cadmium stress in the embodiments of the present invention.

[0023] Among them, A represents the DAB (for detecting H2O2) and NBT (for detecting O2) of wild-type WT, overexpression line SlGRXC9-OE5, and knockout line slgrxc9-1 under 50 μM dCl2 stress at 0d and 5d. -A) Staining phenotypic diagram (scale bar = 1cm); B) Staining comparison diagram of tomato plants under different treatment groups (Control, 50μM CdCl2); C) Quantitative detection results of H2O2 content in tomatoes under cadmium stress (comparison with WT, SlGRXC9-OE1, slgrxc9-CR1); D) Quantitative detection results of MDA (malondialdehyde, reflecting the degree of membrane lipid peroxidation) content in tomatoes under cadmium stress (comparison with WT, SlGRXC9-OE1, slgrxc9-CR1).

[0024] Figure 4 shows the effect of SlGRXC9 on the activity of antioxidant enzymes and cadmium content in tomatoes under cadmium stress in the embodiments of the present invention.

[0025] In this diagram, A shows the results of SOD (superoxide dismutase) activity detection in tomatoes under cadmium stress (compared to WT, SlGRXC9-OE1, and slgrxc9-CR1); B shows the results of POD (peroxidase) activity detection in tomatoes under cadmium stress (compared to WT, SlGRXC9-OE1, and slgrxc9-CR1); and C shows the results of CAT (catalase) activity detection in tomatoes under cadmium stress (compared to WT, SlGRXC9-OE1, and slgrxc9-CR1). D shows the results of GR (glutathione reductase) activity detection in tomatoes under cadmium stress (compared to WT, SlGRXC9-OE1, and slgrxc9-CR1); E shows the results of cadmium content detection in the aboveground parts of tomatoes under cadmium stress (compared to WT, SlGRXC9-OE1, and slgrxc9-CR1); F shows the results of cadmium content detection in the underground parts of tomatoes under cadmium stress (compared to WT, SlGRXC9-OE1, and slgrxc9-CR1). Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0028] Please refer to Figures 1-4. This invention provides an application and method of the SlGRXC9 gene in improving the tolerance of tomatoes to cadmium stress. This gene enhances the activity of the endogenous antioxidant enzyme system in tomatoes, reduces the accumulation of reactive oxygen species and oxidative damage induced by cadmium stress, thereby improving the tolerance of tomatoes to cadmium stress, without affecting the absorption and accumulation of cadmium ions by tomatoes.

[0029] The technical solution of the present invention will be described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental materials, reagents and methods used are all common knowledge in the field of plant genetic engineering. The embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0030] I. Experimental Materials and Reagents

[0031] (a) Biomaterials

[0032] Tomato material: The tomato variety used in this example is Micro-Tom, which is a commonly used model variety in plant gene function research and is easy to cultivate indoors and perform genetic transformation.

[0033] Yeast materials: The cadmium-sensitive yeast mutant Δycf1 was purchased from the YGRC (Saccharomyces cerevisiae Genetic Resource Center); the yeast strain used for the empty vector control was the Δycf1 strain transformed with the empty vector pYES2 (Δycf1+EV).

[0034] Vectors and strains: Plant overexpression vector pCAMBIA1300, yeast expression vector pYES2, and CRISPR / Cas9 backbone vector pYLCRISPR / Cas9-DH are all commonly used vectors in genetic engineering; Agrobacterium strain GV3101 was used for tomato genetic transformation. This strain is a commonly used strain for plant binary vector transformation and has the ability to efficiently infect tomato leaves.

[0035] (II) Main Reagents

[0036] Molecular biology reagents: Trizol reagent (purchased from Invitrogen) was used for RNA extraction; SYBR GreenPCR Master Mix (purchased from Takara) was used for qRT-PCR detection; restriction endonucleases KpnⅠ, EcoRI, and XbaⅠ (purchased from NEB) were used for vector construction; DNA ligase (purchased from Takara) was used for fragment ligation; high-fidelity DNA polymerase (purchased from Vazyme) was used for PCR amplification to ensure the accuracy of the amplified sequences.

[0037] Physiological and biochemical test reagents: Hydrogen peroxide test kit, malondialdehyde (MDA) test kit, and superoxide dismutase (SOD) / peroxidase (POD) / catalase (CAT) / glutathione reductase (GR) activity test kit were all purchased from Nanjing Jiancheng Bioengineering Institute; DAB staining solution and NBT staining solution were purchased from Solarbio.

[0038] Other reagents: CdCl2 was of analytical grade (purchased from Sinopharm Group); yeast culture medium (YPD medium, SD-Ura deficient medium) and tomato tissue culture medium (MS medium) were prepared according to conventional formulas.

[0039] (iii) Sequence Information

[0040] The nucleotide and amino acid sequences involved in this invention are as follows:

[0041] SEQ ID NO.1: Core coding region sequence of SlGRXC9 gene (Solyc07g053550)

[0042] ATGATGCAACAAGCACTTCCTTACAAGTCATCATGTATATCTCTAACACCAAGAGTTGATCGACATCATCGTGTAAGTAATATCAATTCATTATTATACGTTAAAGGTTCAAAAGAAGAATTGAATAACGTTGTTAAGGATAACGCGGTTATCGTTGTGGGAAGACGAGGTTGTTGTATGAGCCATGTTGTGAAACGTTTACTTCATTGTCTCGGAGCAAATCC TGCCATTTATGAAATCGAGGAAGACGATGAAAACGAAGTGGTTGATGAGTTGGAGAATATTATCGTCGCCGGAGGTAGTGATCGGAAAGACACCGGACGGTTGCAATTTCCGGCGGTGTTCGTCGGAGGGGAGCTGTTTGGTGGATTGGATCGGATTATGGCGGCTCATATTACCGGCGAGTTGACTCCTGTGTTGAAAAAGGCTGGAGCCTTATGGCTTTGA;

[0043] SEQ ID NO.2: Protein sequence encoded by the SlGRXC9 gene

[0044] MMQQALPYKSSCISLTPRVDRHHRVSNINSLLYVKGSKEELNNVVKDNAVIVVGRRGCCMSHVVKRLLHCLGANPAIYEIEEDDENEVVDELENIIVAGGSDRKDTGRLQFPAVFVGGELFGGLDRIMAAHITGELTPVLKKAGALWL;

[0045] SEQ ID NO.3: qRT-PCR upstream primer (qRT-C9-F)

[0046] GTTGTTGTATGAGCCATGTTGT;

[0047] SEQ ID NO.4: qRT-PCR downstream primer (qRT-C9-R)

[0048] CAACTCATCAACCACTTCGTTT;

[0049] SEQ ID NO.5: Upstream primer for yeast expression vector construction (yesScT7-F)

[0050] cgacgatgacgataaggtacctATGATGCAACAAGCACTTCC;

[0051] SEQ ID NO.6: Downstream primer for yeast expression vector construction (yesScT7-R)

[0052] tgctggatatctgcagaattcTCAAAGCCATAAGGCTCCAG;

[0053] SEQ ID NO.7: Upstream primer for constructing the tomato overexpression vector (OE-ScT7-F)

[0054] atttggagaggacagggtaccATGATGCAACAAGCACTTCC;

[0055] SEQ ID NO.8: Downstream primer for constructing the tomato overexpression vector (OE-ScT7-R)

[0056] cgaactagtgtcgactctagaAAGCCATAAGGCTCCAGC;

[0057] SEQ ID NO.9: CRISPR / Cas9 knockout target sequence (ScT-SG)

[0058] CGATGATGTCGATCAACTCT;

[0059] SEQ ID NO.10: Upstream primer of the tomato Actin gene (Actin-F)

[0060] TGGCATCACACTTTCTACAACGA;

[0061] SEQ ID NO.11: Downstream primer of the tomato Actin gene (Actin-R)

[0062] GGATGGCTGGAAGAGTGATAAAC;

[0063] SEQ ID NO.12: Upstream primer for identifying the target region of SlGRXC9 CRISPR / Cas9 knockout strain

[0064] GATGATGTCGATCAACTCTGG;

[0065] SEQ ID NO.13: Downstream primers for identifying the target region of SlGRXC9 CRISPR / Cas9 knockout strains

[0066] CCTTGTAGAGATATACATGAT.

[0067] II. Specific implementation methods are as follows:

[0068] Example 1: Expression characteristics of SlGRXC9 gene under cadmium stress

[0069] 1. Tomato seedling culture: Select plump Micro-Tom tomato seeds, disinfect them with 5% sodium hypochlorite solution for 10 minutes, rinse them 3-5 times with sterile water, sow them in Petri dishes containing MS medium, and place them in an artificial climate chamber for cultivation. The cultivation conditions are: light intensity 200 μmol·m⁻². -2 ·s -1 The seedlings were kept under a 16-hour light-to-dark cycle, with a temperature of 25°C and a relative humidity of 70%. Once the seedlings had two leaves and a central bud, they were transplanted into pots containing a sterilized substrate (peat soil: perlite = 3:1) for further cultivation.

[0070] 2. Cadmium stress treatment: When tomato seedlings reached the four-leaf stage, seedlings with uniform growth were selected and divided into two groups. The control group was irrigated with Hoagland nutrient solution without CdCl2, while the treatment group was irrigated with Hoagland nutrient solution containing 50 μM CdCl2. Tomato root tissues were collected at 0, 1, 2, 3, 4, and 5 days of treatment for the detection of SlGRXC9 gene expression. At the same time, young leaves, green-ripe fruits, color-changing fruits, and red-ripe fruits of tomatoes under normal growth conditions were collected for tissue-specific expression analysis.

[0071] 3. RNA extraction and cDNA synthesis: Total RNA was extracted from each tissue sample using Trizol reagent. The integrity and purity of the RNA were verified by 1% agarose gel electrophoresis and Nanodrop nucleic acid detection instrument to ensure that the RNA was not degraded and met the purity standard (A260 / A280 ratio between 1.8 and 2.0). Using the extracted total RNA as a template, the first strand of cDNA was synthesized according to the reverse transcription kit (purchased from Takara).

[0072] 4. qRT-PCR detection: Using cDNA as a template, qRT-PCR amplification was performed using primers shown in SEQ ID NO.3 and SEQ ID NO.4, with the tomato Actin gene as an internal reference gene (primer sequences are SEQ ID NO.10 and SEQ ID NO.11). The amplification program was: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing for 30 s, for 40 cycles; melting curve analysis: 95℃ for 15 s, 60℃ for 1 min, 95℃ for 15 s.

[0073] Each sample was configured with 3 biological replicates and 3 technical replicates, using 2 - The relative expression level of the SlGRXC9 gene was calculated using the ΔΔCt method.

[0074] qRT-PCR results (Figure 1A) showed that under 50 μM CdCl2 stress, the relative expression level of the SlGRXC9 gene in tomato roots gradually increased with treatment time, showing a significant upregulation trend within 1-4 days of treatment, with the highest expression level reaching about 10 times that of the control group, indicating that the expression of the SlGRXC9 gene was significantly induced by cadmium stress. Tissue-specific expression analysis (Figure 1B) showed that the SlGRXC9 gene had the highest expression level in tomato fruits during the color-changing and red-ripe stages, followed by the root system, and the expression level was low in young leaves, indicating that this gene has a preferential expression characteristic in tomato reproductive organs and absorptive organs.

[0075] The above results confirm that the SlGRXC9 gene is closely related to the tomato cadmium stress response, providing a theoretical basis for subsequent gene function verification.

[0076] Example 2: Verification of the cadmium tolerance function of the SlGRXC9 gene in yeast

[0077] 1. Amplification of the SlGRXC9 gene coding sequence: Using the tomato root cDNA synthesized in Example 1 as a template, PCR amplification was performed using primers shown in SEQ ID NO. 5 and SEQ ID NO. 6. KpnⅠ and EcoRI restriction sites were introduced upstream and downstream of the primers, respectively (where the uppercase letters represent the protein coding region-specific sequence of SlGRXC9, and the lowercase letters represent the introduced restriction sites and vector homologous sequences). The amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles; 72℃ final extension for 10 min. After PCR product detection by 1% agarose gel electrophoresis, the target fragment was recovered using a gel extraction kit (purchased from Axygen).

[0078] 2. Construction of yeast expression vector: The recovered SlGRXC9 gene coding region fragment and the yeast expression vector pYES2 were double-digested with KpnⅠ and EcoRI, respectively. After gel recovery, the digestion products were ligated overnight at 16℃ using T4 DNA ligase to construct the recombinant expression vector pYES2-SlGRXC9. The ligation product was transformed into E. coli DH5α competent cells (purchased from Takara), plated on LB agar plates containing ampicillin (100 μg / mL), and incubated at 37℃ for 12-16 h. Single colonies were picked for PCR identification and sequencing verification to ensure that the target gene was correctly inserted into the vector.

[0079] 3. Yeast Transformation: The recombinant vector pYES2-SlGRXC9 and the empty vector pYES2 were transformed into competent yeast cadmium-sensitive mutant Δycf1 cells using the lithium acetate transformation method. The specific steps are as follows:

[0080] The Δycf1 yeast strain was inoculated into YPD medium and cultured at 30°C with shaking at 200 rpm until OD600 = 0.5-1.0. The cells were collected by centrifugation, washed twice with sterile water, resuspended in transformation buffer (0.1M LiAc, 10mM DTT, 0.6M sorbitol), and incubated at 30°C for 30 min. Vector DNA (5 μg) and salmon sperm DNA (10 μg) were added, and the cells were incubated at 30°C for 30 min, followed by heat shock at 42°C for 15 min. The cells were collected by centrifugation, resuspended in YPD medium, and cultured at 30°C with shaking for 2 h. The bacterial culture was spread on SD-Ura deficient medium plates and cultured at 30°C for 3-4 days. Positive clones were picked to obtain the Δycf1+SlGRXC9 and Δycf1+EV strains.

[0081] 4. Yeast Cadmium Tolerance Test: Δycf1+SlGRXC9 and Δycf1+EV strains were inoculated into SD-Ura liquid medium and cultured at 30℃ with shaking at 200 rpm until OD600 = 1.0. The bacterial suspension was then diluted with sterile water to OD600 = 0.1, followed by a 10-fold serial dilution (10... -1 10 -2 10 -3 10 -4 ); 10 μL of diluted bacterial suspension was added dropwise to SD-Ura solid medium plates containing 0 μM, 15 μM, 25 μM, and 50 μM CdCl2, respectively, and incubated at 30℃ for 3-5 days. The yeast growth was observed and photographed. At the same time, the above two strains were inoculated into SD-Ura liquid medium containing 25 μM CdCl2 and cultured at 30℃ with shaking at 200 rpm. The OD600 value of the bacterial suspension was measured at 0 h, 10 h, 20 h, 30 h, and 40 h, and growth curves were plotted.

[0082] 5. Detection of cadmium content in yeast cells: Δycf1+SlGRXC9 and Δycf1+EV strains were inoculated into SD-Ura liquid medium containing 25 μM dCl2 and cultured at 30℃ and 200 rpm for 24 h with shaking. The cells were collected by centrifugation, washed three times with sterile water, freeze-dried, and weighed. The cell samples were digested with concentrated nitric acid using microwave digestion, and the cadmium content was determined by inductively coupled plasma mass spectrometry (ICP-MS, Thermo Fisher Scientific). Three replicates were set up for each sample.

[0083] The results of yeast growth phenotypic observation (Figure 1C, Figure 1D) showed that there was no significant difference in growth between the Δycf1+SlGRXC9 strain and the Δycf1+EV strain on CdCl2-free medium. On media containing 15μM, 25μM, and 50μM CdCl2, the growth capacity and survival rate of the Δycf1+SlGRXC9 strain were significantly higher than those of the Δycf1+EV strain, and this difference became more pronounced with increasing CdCl2 concentration. The growth curve results showed that under 25μM CdCl2 stress, the OD600 value of the Δycf1+SlGRXC9 strain was consistently higher than that of the Δycf1+EV strain, indicating that heterologous expression of the SlGRXC9 gene can significantly enhance the yeast's tolerance to cadmium stress. The cadmium content detection results (Figure 1E) showed that there was no significant difference in cadmium content between the Δycf1+SlGRXC9 strain and the Δycf1+EV strain, indicating that the mechanism by which the SlGRXC9 gene enhances yeast cadmium tolerance does not affect cadmium absorption and accumulation, which is consistent with the technical principle of this invention.

[0084] Example 3: Construction of SlGRXC9 transgenic tomato and phenotypic analysis under cadmium stress

[0085] 1. Construction of tomato overexpression vector: Using tomato root cDNA as a template, PCR amplification was performed using primers shown in SEQ ID NO.7 and SEQ ID NO.8. KpnⅠ and XbaⅠ restriction enzyme sites were introduced upstream and downstream of the primers, respectively (where the uppercase letters are the protein coding region-specific sequences of SlGRXC9, and the lowercase letters are the introduced restriction enzyme sites and vector homologous sequences). The amplification procedure was the same as in Example 2. The target fragment was recovered and double-digested with the plant expression vector pCAMBIA1300. The digested products were ligated and transformed into Escherichia coli DH5α competent cells. The recombinant overexpression vector pCAMBIA1300-SlGRXC9 was obtained by PCR identification and sequencing verification.

[0086] 2. Construction of CRISPR / Cas9 knockout vector: Using the sequence shown in SEQ ID NO.9 as the target, CRISPR / Cas9 knockout primers were designed and constructed according to the pYLCRISPR / Cas9-DH vector instructions. The target sequence was inserted downstream of the U6-26 promoter of the vector to construct the recombinant knockout vector pYLCRISPR / Cas9-SlGRXC9. After transformation into E. coli DH5α, sequencing verification was performed.

[0087] 3. Tomato genetic transformation: Agrobacterium-mediated leaf disc transformation was used. The specific steps are as follows:

[0088] The recombinant overexpression vector pCAMBIA1300-SlGRXC9 and the knockout vector pYLCRISPR / Cas9-SlGRXC9 were transformed into Agrobacterium GV3101 competent cells, respectively. Positive Agrobacterium colonies were picked and inoculated into LB liquid medium containing rifampicin (50 μg / mL) and kanamycin (50 μg / mL), and cultured at 28°C with shaking at 200 rpm until OD600 = 0.5-0.6. Leaves from 'Micro-Tom' aseptic tomato seedlings were cut into 0.5cm × 0.5cm leaf discs and immersed in Agrobacterium bacterial solution for 10 min, gently shaking during this time. The leaf discs were then removed and sterilized using sterile... Blot the surface bacterial solution with filter paper and inoculate it onto co-culture medium (MS + 2 mg / L 6-BA + 0.2 mg / L LIAA) and incubate in the dark at 25°C for 2 days. Transfer the leaf discs to selection medium (MS + 2 mg / L 6-BA + 0.2 mg / L LIAA + 50 μg / mL hygromycin + 200 μg / mL cephalosporin) and culture under light. Change the medium every 2 weeks until adventitious shoots differentiate. Cut off the adventitious shoots and transfer them to rooting medium (1 / 2 MS + 0.1 mg / L LIAA + 50 μg / mL hygromycin + 200 μg / mL cephalosporin) for culture. After rooting, transgenic tomato seedlings are obtained and transplanted into the substrate for culture.

[0089] 4. Identification of transgenic lines:

[0090] Identification of overexpression lines: Total RNA was extracted from the leaves of transgenic tomatoes and cDNA was synthesized by reverse transcription. The relative expression level of the SlGRXC9 gene was detected by the qRT-PCR method in Example 1. Wild-type (WT) tomatoes were used as controls to screen for lines with significantly upregulated expression of the SlGRXC9 gene.

[0091] Identification of knockout lines: Genomic DNA was extracted from transgenic tomato leaves, and identification primers were designed upstream and downstream of the target sequence (upstream is SEQ ID NO.12; downstream is SEQ ID NO.13). The target region was amplified by PCR, and the amplified products were sequenced and compared with the wild-type sequence to screen homozygous mutant lines.

[0092] 5. Phenotypic analysis under cadmium stress: Correctly identified overexpression lines (OE5, SlGRXC9 gene upregulated approximately 9-fold), knockout lines (slgrxc9-1, homozygous insertion mutation), and wild-type (WT) tomato seedlings were selected and transplanted into pots containing sterilized substrate. When the seedlings reached the four-leaf-one-heart stage, they were divided into control and treatment groups. The control group was watered with Hoagland nutrient solution without CdCl2, while the treatment group was watered with Hoagland nutrient solution containing 50 μM CdCl2. After 2 weeks of treatment, the aboveground dry weight, underground dry weight, chlorophyll a content, and chlorophyll b content of each line were measured, with 3 biological replicates for each indicator.

[0093] The chlorophyll content was determined by ethanol extraction: 0.1g of leaf sample was taken, 5mL of 80% ethanol was added, and the sample was soaked in the dark for 24h. The absorbance values ​​at 663nm and 645nm were measured respectively, and the contents of chlorophyll a and chlorophyll b were calculated according to the formula.

[0094] The results of transgenic line identification showed that six independent SlGRXC9 overexpression lines were successfully obtained. Among them, the OE5 line had the highest relative expression level of SlGRXC9 gene, which was about 9 times that of wild type (Figure 2A). One SlGRXC9 homozygous knockout line slgrxc9-1 was successfully obtained. This line had a one-base insertion at the target sequence, which caused the gene coding frame to shift (Figure 2B, C).

[0095] Phenotypic analysis showed that under normal growth conditions, there were no significant differences in aboveground dry weight, underground dry weight, chlorophyll a content, and chlorophyll b content between the overexpressing line OE5, the knockout line slgrxc9-1, and the wild-type WT. After treatment with 50 μM CdCl2 stress, the growth of all three plants was significantly inhibited, and the above growth indicators and photosynthetic pigment content all decreased significantly, but the degree of inhibition differed significantly: the aboveground dry weight, underground dry weight, chlorophyll a content, and chlorophyll b content of the overexpressing line OE5 were significantly higher than those of WT, while the above indicators of the knockout line slgrxc9-1 were significantly lower than those of WT (Figures 2E, 2F, 2G, and 2H). This indicates that overexpression of the SlGRXC9 gene can significantly alleviate the inhibitory effect of cadmium stress on tomato growth, while gene knockout exacerbates the damage caused by cadmium stress, confirming that the SlGRXC9 gene plays a crucial role in improving tomato tolerance to cadmium stress.

[0096] Example 4: Regulatory effect of SlGRXC9 gene on oxidative stress in tomato

[0097] 1. Cadmium stress treatment: Overexpression line OE5, knockout line slgrxc9-1 and wild-type WT tomato seedlings were selected and cultured to the four-leaf-one-heart stage. The treatment group was watered with Hoagland nutrient solution containing 50 μM CdCl2, while the control group was watered with Hoagland nutrient solution without CdCl2. Leaf samples were collected 2 weeks after treatment for subsequent testing.

[0098] 2. Detection of Reactive Oxygen Species (ROS) Accumulation:

[0099] DAB staining for H2O2 detection: Take fresh leaves, soak them in DAB staining solution, incubate at 25°C for 8 hours in the dark, decolorize with 75% ethanol until the leaves turn white, observe the staining depth of the leaves and take pictures to record.

[0100] NBT staining detection of O2 - Take fresh leaves, soak them in NBT staining solution, incubate at 25°C for 6 hours in the dark, decolorize by boiling with 75% ethanol, observe the staining depth of the leaves and take pictures to record.

[0101] Quantitative analysis of H2O2 content: The H2O2 content in leaf samples was determined using a hydrogen peroxide detection kit, following the instructions in the manual.

[0102] 3. Detection of membrane lipid peroxidation damage: The content of MDA in leaf samples was determined using an MDA detection kit. The MDA content reflects the degree of membrane lipid peroxidation damage.

[0103] 4. Antioxidant enzyme activity detection: Using the corresponding detection kits, the activities of SOD (superoxide dismutase), POD (peroxidase), CAT (catalase) and GR (glutathione reductase) in leaf samples were measured respectively. The operation steps were strictly followed according to the kit instructions.

[0104] 5. Cadmium content detection in tomato plants: Aboveground and underground tissues of each plant line after cadmium stress treatment were collected, cleaned, freeze-dried, weighed, and digested with concentrated nitric acid using microwave. The cadmium content was determined by ICP-MS, with three replicates for each sample.

[0105] Reactive oxygen species (ROS) detection results showed that under 50 μM CdCl2 stress, the DAB and NBT staining of leaves from the overexpressing line OE5 was significantly lighter than that of the wild-type WT, while the staining of the knockout line slgrxc9-1 was significantly darker than that of WT (Fig. 3A, Fig. 3B). Quantitative H2O2 content results (Fig. 3C) indicated that the H2O2 content of the OE5 line was significantly lower than that of WT, while the H2O2 content of the slgrxc9-1 line was significantly higher than that of WT, confirming that the SlGRXC9 gene can significantly reduce cadmium stress-induced ROS accumulation. MDA content detection results (Fig. 3D) showed that the MDA content of the OE5 line was significantly lower than that of WT, while the MDA content of the slgrxc9-1 line was significantly higher than that of WT, indicating that the SlGRXC9 gene can effectively alleviate cadmium stress-induced membrane lipid peroxidation damage. The results of antioxidant enzyme activity detection (Figures 4A-4D) showed that under cadmium stress, the activities of SOD, POD, CAT, and GR in the OE5 strain were significantly higher than those in the WT strain, while the activities of the above-mentioned antioxidant enzymes in the slgrxc9-1 strain were significantly lower than those in the WT strain, confirming that the SlGRXC9 gene can enhance the activity of the antioxidant enzyme system in tomatoes. The results of cadmium content detection (Figures 4E and 4F) showed that there was no significant difference in cadmium content between OE5, slgrxc9-1, and WT in either the aboveground or underground parts, indicating that the mechanism by which the SlGRXC9 gene improves tomato tolerance to cadmium stress is through enhancing antioxidant capacity, rather than altering cadmium absorption and accumulation, which is consistent with the technical solution of this invention.

[0106] In summary, this invention clarifies the function of the SlGRXC9 gene and provides its application and related methods in improving the tolerance of tomatoes to cadmium stress. This technical solution does not affect the absorption of essential micronutrients by tomatoes, has high genetic stability, and is low in cost. It can be widely applied to the safe production of tomatoes and the creation of stress-resistant germplasm under cadmium-contaminated soil conditions, and has important agricultural application value.

[0107] It should be noted that, although this specification describes the embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. An SlGRXC9 gene, characterized in that, The core coding region sequence of the gene is SEQ ID NO.1, and the gene number is Solyc07g053550.

2. The SlGRXC9 gene according to claim 1, characterized in that, The protein sequence encoded by the gene is SEQ ID NO.

2.

3. A method for improving tomato tolerance to cadmium stress using the SlGRXC9 gene, characterized in that, By enhancing the expression or activity of the SlGRXC9 gene as described in claim 1 in tomatoes, the activity of the endogenous antioxidant enzyme system in tomatoes is enhanced, and the accumulation of reactive oxygen species and membrane lipid peroxidation damage induced by cadmium stress are reduced, thereby improving the tolerance of tomatoes to cadmium stress.

4. The method for improving tomato cadmium stress tolerance using the SlGRXC9 gene according to claim 3, characterized in that, The method for enhancing SlGRXC9 gene expression is to construct an SlGRXC9 gene overexpression vector, introduce the overexpression vector into tomato cells or tissues, and obtain transgenic tomatoes that overexpress the SlGRXC9 gene.

5. The method for improving tomato cadmium stress tolerance using the SlGRXC9 gene according to claim 3, characterized in that, The overexpression vector was introduced into tomato cells or tissues using an Agrobacterium-mediated transformation method.

6. The method for improving tomato cadmium stress tolerance using the SlGRXC9 gene according to claim 3, characterized in that, The endogenous antioxidant enzyme system includes superoxide dismutase, peroxidase, catalase, and glutathione reductase.

7. The method for improving tomato cadmium stress tolerance using the SlGRXC9 gene according to claim 3, characterized in that, The method described does not affect the absorption and accumulation of cadmium ions by tomatoes.

8. The application of the SlGRXC9 gene as described in claim 1 in improving tomato tolerance to cadmium stress, characterized in that, The SlGRXC9 gene can be used for the safe production of tomatoes in cadmium-contaminated soil or for the creation of cadmium-tolerant tomato germplasm.

9. The application of the SlGRXC9 gene according to claim 8 in improving tomato tolerance to cadmium stress, characterized in that, The application method involves using the SlGRXC9 gene as a molecular breeding target to screen or cultivate tomato varieties resistant to cadmium stress.

10. The application of the SlGRXC9 gene according to claim 8 in improving tomato tolerance to cadmium stress, characterized in that, The application method is to plant genetically modified tomatoes obtained by the method described in claim 5.

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