Application of SlJMJ524 gene in improvement of cadmium resistance of tomato

Abstract

The invention discloses application of an SlJMJ524 gene in improvement of cadmium resistance of tomatoes, and belongs to the technical field of plant stress resistance. The invention aims to provide a method for resisting cadmium stress of tomatoes. The invention provides application of SlJMJ524 protein in improvement of cadmium resistance of tomatoes. The amino acid sequence of the SlJMJ524 protein is as shown in SEQ ID NO.24. The invention also provides application of the SlJMJ524 protein in improvement of cadmium resistance of tomatoes. The SlJMJ524 gene can enhance the resistance of tomatoes to cadmium stress, and a theoretical basis is provided for cadmium resistance of plants.

Description

Technical Field

[0001] The invention belongs to the technical field of plant stress resistance, and particularly relates to application of SlJMJ524 gene in improving cadmium resistance of tomatoes. Background Art

[0002] With the rapid development of economy and highly intensive agricultural production, soil pollution is becoming increasingly serious. Nearly 1 / 5 of the soil in my country is seriously polluted. Various human production processes, such as mining, smelting, metal electroplating, sewage sludge treatment, and the application of pesticides and fertilizers, disperse a large amount of heavy metals into the environment, seriously destroying the balance of the ecosystem. In my country, the rate of heavy metal exceeding the standard in cultivated land accounts for about 19.4% of the country, and the rate of soil Cd exceeding the standard is 7.0%. Today, 2.786×109m2 of agricultural soil is contaminated by Cd.

[0003] The World Health Organization (WHO) considers Cd to be a food contaminant second only to aflatoxin and inorganic arsenic in terms of lethality. Cd is persistently toxic, non-biodegradable, bioaccumulative, and highly soluble. Cd can affect plant growth and development, cause cell membrane lipid peroxidation, and alter the antioxidant system. In addition, due to its strong migration ability and easy absorption by plants, Cd can be transferred through the food chain and accumulated in the human body. In particular, the consumption of "cadmium rice" and "cadmium vegetables" has increased the health risks of Cd to the human body.

[0004] Tomato, as an important vegetable crop worldwide, has a direct impact on people's food safety due to its quality. Tomato is one of the vegetables that easily absorbs and accumulates cadmium. Under 2.0 mg / kg Cd stress, the cadmium exceeding the standard rate in the edible parts of all tested varieties of tomatoes is 100%, and excessive accumulation of cadmium in tomatoes may pose a greater threat to human health. Therefore, finding a method for tomato to tolerate cadmium stress is a technical problem that needs to be solved urgently. Summary of the invention

[0005] The purpose of the invention is to provide a method for tomato to resist cadmium stress.

[0006] The present invention provides an application of a SlJMJ524 protein in improving the cadmium resistance of tomatoes. The amino acid sequence of the SlJMJ524 protein is shown in SEQ ID NO.24.

[0007] The invention provides an application of a SlJMJ524 gene in improving the cadmium resistance of tomatoes, wherein the nucleic acid molecule of the SlJMJ524 gene is shown as SEQ ID NO.3.

[0008] The invention provides an application of a recombinant vector containing a SlJMJ524 gene in improving the cadmium resistance of tomatoes. The nucleic acid molecule of the SlJMJ524 gene is shown in SEQ ID NO.3.

[0009] It is further defined that the starting vector of the recombinant vector is pCAMBIA2300.

[0010] The invention provides an application of a recombinant microbial cell containing a SlJMJ524 gene in improving the cadmium resistance of tomatoes. The nucleic acid molecule of the SlJMJ524 gene is shown in SEQ ID NO.3.

[0011] It is further defined that the recombinant microbial cell is a eukaryotic microbial cell or a prokaryotic microbial cell.

[0012] The present invention provides an application of a tomato overexpressing a SlJMJ524 gene in improving cadmium resistance. The nucleic acid molecule of the SlJMJ524 gene is shown in SEQ ID NO.3.

[0013] The present invention provides a method for improving cadmium resistance of tomatoes, characterized in that the specific steps of the method are as follows:

[0014] Step 1: Connect the SlJMJ524 gene to the pCAMBIA2300 vector to obtain a recombinant vector;

[0015] Step 2: Transform the recombinant vector obtained in step 1 into Agrobacterium to obtain recombinant Agrobacterium;

[0016] Step 3: Infect tomatoes with the recombinant Agrobacterium obtained in step 2 to obtain transgenic tomatoes.

[0017] It is further defined that the primers for amplifying the SlJMJ524 gene in step 1 are SEQ ID NO.1 and SEQ ID NO.2.

[0018] It is further defined that the nucleic acid molecule of the SlJMJ524 gene in step 1 is as shown in SEQ ID NO.3.

[0019] Beneficial effects: The present invention discovered a tomato cadmium-resistant gene SlJMJ524. The experiments of the present invention have shown that overexpressing the gene in tomatoes can enhance the tolerance of tomatoes to cadmium stress at the seedling stage, indicating that the gene can lay a foundation for the research of cultivating transgenic plants with cadmium resistance. Through Agrobacterium-mediated tomato homologous transformation, a tomato strain with the SlJMJ524 gene was successfully obtained. After cadmium stress treatment, the phenotype and related physiological indicators were analyzed. The results showed that compared with the control, the overexpression strain of the SlJMJ524 gene tomato had a better phenotype, larger root length and plant height values; lower MDA content, and higher activities of antioxidant enzymes such as SOD, POD, and CAT. The expression of marker genes related to cadmium stress in transgenic tomatoes was analyzed. It was found that under cadmium stress, stress marker genes such as APX1, ABCC2, and POD were upregulated in SlJMJ524 overexpressing transgenic tomatoes, and the expression levels were significantly higher than those of WT. Therefore, we speculate that the SlJMJ524 gene may enhance tolerance to cadmium stress by regulating the expression levels of stress-induced marker genes. Such research results confirm that the SlJMJ524 gene can enhance the resistance of tomatoes to cadmium stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Molecular identification of SlJMJ524 transgenic tomato overexpression plants. WT is wild type, OE1-9 is SlJMJ524 overexpression line #1-9;

[0021] Figure 2 Molecular identification of SlJMJ524 transgenic tomato gene knockout plants. M is DL 2000 marker; negative control PCR template is water; positive control template is SlJMJ524 gene knockout plasmid; CP1 and CP2 are SlJMJ524 gene knockout strain #1 and SlJMJ524 gene knockout strain #2;

[0022] Figure 3 The plant phenotype, plant height and root length of SlJMJ524 transgenic tomatoes under cadmium stress. A, plant phenotype of SlJMJ524 transgenic tomatoes under cadmium stress; B, plant height and root length of each line of SlJMJ524 transgenic tomatoes under normal conditions; C, plant height and root length of each line of SlJMJ524 transgenic tomatoes under cadmium stress. Among them, WT is the wild type, OE2 and OE8 are #2 and SlJMJ524 overexpression line #8, CP1 and CP2 are SlJMJ524 gene knockout line #1 and SlJMJ524 gene knockout line #2;

[0023] Figure 4These are the physiological response indicators of SlJMJ524 transgenic tomatoes under cadmium stress. AD, determination of POD activity, SOD activity, CAT activity and malondialdehyde content in SlJMJ524 transgenic tomatoes. EH, determination of photosynthetic pigment content in SlJMJ524 transgenic tomatoes; I, DAB and NBT staining of SlJMJ524 transgenic tomatoes; J, determination of hydrogen peroxide content in SlJMJ524 transgenic tomatoes; K, determination of superoxide anion content in SlJMJ524 transgenic tomatoes. Among them, WT is the wild type, SlJMJ524-OE2 and SlJMJ524-OE8 are SlJMJ524 overexpression strain #2 and SlJMJ524 overexpression strain #8, SlJMJ524-CP1 and SlJMJ524-CP2 are SlJMJ524 gene knockout strain #1 and SlJMJ524 gene knockout strain #2; Figure 5 Expression analysis of marker genes related to cadmium stress. WT is wild type, OE2 and OE8 are SlJMJ524 overexpression strain #2 and SlJMJ524 overexpression strain #8, CP1 and CP2 are SlJMJ524 gene knockout strain #1 and SlJMJ524 gene knockout strain #2. DETAILED DESCRIPTION

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0026] The DH5α competent cells and Agrobacterium GV3101 competent cells in the following examples are products of Quanshijin Company.

[0027] Example 1. Cloning of the tomato S1JMJ524 gene

[0028] 1. Processing of plant materials

[0029] Select plump Micro-Tom tomato seeds, sterilize them in a solution containing 2% sodium hypochlorite for 10 minutes, pour out the sodium hypochlorite, rinse with sterile water 6-7 times, place them on moist filter paper, and culture them in the dark at 25℃ for 4 days to germinate. When the buds grow to about 1-2 cm, transfer them to a nutrient pot filled with vermiculite and peat soil in a ratio of 1:1, and place them in a greenhouse for cultivation. The growth conditions in the greenhouse are 25℃ / 20℃ (day / night) and the photoperiod is 16 hours of light / 8 hours of darkness. When the seedlings grow to four leaves and one heart, take the leaves and put them in an EP tube and store them at -80℃.

[0030] 2. RNA Extraction

[0031] The total RNA from the leaves of the four-leaf, one-heart tomato was extracted using the Plant Total RNA Isolation Kit (Foregene).

[0032] 3. Obtaining cDNA

[0033] The total RNA was used as a template for reverse transcription to obtain cDNA.

[0034] 4. PCR amplification

[0035] Using the above cDNA as a template, Primer-KS and Primer-KAS primers were used for PCR amplification to obtain PCR amplification products. The primer sequences are as follows:

[0036] Primer-KS: 5'-GAACACAACTGCATTCCACT-3' (SEQ ID NO. 1);

[0037] Primer-KAS: 5'-GTATAACGAGACGAGACCG-3' (SEQ ID NO. 2).

[0038] PCR amplification system (12.5 μl): cDNA 0.5 μl, Primer-F 0.5 μl, Primer-R 0.5 μl, Mix enzyme 6.25 μl, ddH2O 4.75 μl.

[0039] PCR amplification conditions: 94°C for 2 min, 94°C for 15 s, 57°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 10 min; terminate the reaction at 4°C.

[0040] The PCR amplification product was subjected to 1% agarose gel electrophoresis to obtain a band with a molecular weight of approximately 1.3 Kb, and the PCR amplification product was recovered using an agarose gel extraction kit (Omega Gel Extraction kit); it was connected to the pCAMBIA-1300 vector to obtain a recombinant plasmid, which was named pCAMBIA1300-SlJMJ524, and was transformed into Escherichia coli DH5α competent cells and then sent for sequencing.

[0041] The sequencing results showed that the PCR amplification obtained an amplification product of 1254 bp in size, which was named SlJMJ524 gene, and its nucleotide sequence was shown in SEQ ID NO.3.

[0042] S1JMJ524 gene sequence (SEQ ID NO.3):

[0043]

[0044] S1JMJ524 protein sequence (SEQ ID NO.24):

[0045] MSTSAADAVAGEITGDDDGVLETPFLDSERWNILQSVTEEGGYAYATMTSLAAAGDIRAAEAAKEMAWEQLHSGPWHSVVPIWRDAYSMACMHLAKLHYANGEFNEAIKALDMGIIMGGMMLRDELNLAIRKASEKEKAKSDLGEKGESEIKFVSQEINVAEVQQSLPIKSLSCKLVGKRPALSLEAFLREHYLSGTPVVIRDSMDHWP AKSRWNDMNYLRRVAGFRTVPVEVGKNYLCPEWKQELITFSEFLERIRSNDTTSVETTYLAQHQLFDQIQELQQDIVIPDYCFTGGGEIRSLNAWFGPAGTVTP LHHDPHHNILAQVVGKKYVRLYSASLSDELHPHSETMLSNSSQVDLDNIDEKEFPNILDLEFQDCILEEGEMLYIPPKWWHYVRSLTTSFSVSFWWSDAEKLDD.

[0046] Example 2. Obtaining tomato plants overexpressing SlJMJ524 and phenotypic analysis and physiological index analysis under cadmium stress I. Obtaining tomato plants overexpressing SlJMJ524

[0047] 1. Acquisition of SlJMJ524 gene

[0048] Using pCAMBIA1300-SlJMJ524 obtained in step 4 of Example 1 as a template, PCR amplification was performed using primers Primer-ES and Primer-EAS to obtain a PCR amplification product, namely the SlJMJ524 gene. The primer sequences are as follows:

[0049] Primer-ES: 5'-GAACACAACTGCATTCCACT-3' (SEQ ID NO.4);

[0050] Primer-EAS: 5'-GTATAACGAGACGAGACCG-3' (SEQ ID NO. 5).

[0051] PCR amplification system (12.5 μl): cDNA 0.5 μl, Primer-F 0.5 μl, Primer-R 0.5 μl, Mix enzyme 6.25 μl, ddH2O 4.75 μl.

[0052] PCR amplification conditions: 94°C for 2 min, 94°C for 15 s, 57°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 10 min; terminate the reaction at 4°C.

[0053] 2. Obtaining plant expression vector

[0054] The pCAMBIA 1300 vector and the PCR amplification product were digested with restriction endonucleases and ligated to obtain the recombinant vector pCAMBIA 1300-S1JMJ524, which was then sequenced and verified.

[0055] The sequencing results showed that the recombinant vector pCAMBIA 1300-SlJMJ524 was obtained by replacing the DNA fragment between the restriction sites of the pCAMBIA 1300 vector with the SlJMJ524 gene shown in sequence 1 in the sequence table, while keeping the other sequences of the pCAMBIA1300 vector unchanged.

[0056] 3. Conversion

[0057] The recombinant vector pCAMBIA 1300-SlJMJ524 was transformed into Agrobacterium tumefaciens GV3101 by freeze-thaw method, and positive transformants (transformants containing the SlJMJ524 gene shown in sequence 1 in the sequence table) were obtained by PCR identification and used to infect tomato plants.

[0058] 4. Obtaining the SlJMJ524 tomato overexpression line

[0059] Agrobacterium containing the recombinant vector pCAMBIA1300-SlJMJ524 was used to infect tomato (Micro-Tom) by Agrobacterium-mediated method. Take plump tomato (Micro-Tom) seeds and soak them at 55℃ for 15min, disinfect them with 75% ethanol for 30s, rinse them with sterile water 2-3 times, disinfect them with 2% sodium hypochlorite solution for 15min, rinse them with sterile water 6-7 times, dry them and inoculate them on 1 / 2MS culture medium, place them in the tissue culture room for dark culture, and place them under normal light culture conditions (16h light / 8h dark) for culture when white spots appear on the seeds. After the cotyledons of the sterile seedlings are flattened, the cotyledons are cut into 0.5 cm × 0.5 cm sizes and placed on MS culture medium for culture. The tomato cotyledons are infected with Agrobacterium containing the recombinant vector pCAMBIA 1300-SlJMJ524. Two days later, the infected cotyledons are transferred to a screening medium containing 50 mg / L kanamycin (Kanamycin Sulfate, Biotopped, K6020) until callus sprouts. When the adventitious buds grow to about 2-3 cm, they are cut off and inoculated into a rooting medium. After rooting, they are transferred to a nutrient pot containing vermiculite and peat soil in a ratio of 1:1 to obtain T0 generation SlJMJ524 tomato seeds. After the surface of the tomato seeds is disinfected, they are sown on 1 / 2MS culture medium containing 50 mg / L kanamycin (Kanamycin Sulfate, Biotopped, K6020) for screening to obtain T1 generation SlJMJ524 tomato seedlings. This process was repeated until a T2 generation S1JMJ524 tomato homozygous strain was obtained.

[0060] Extract genomic RNA from T2-generation SlJMJ524 transgenic tomato seedlings and perform qRT-PCR identification. The specific steps are as follows:

[0061] The total RNA of the T2-generation SlJMJ524 tomato plants obtained in step 1 was extracted and reverse transcribed to obtain cDNA; using the cDNA as a template, the Primer-qS and Primer-qAS primer pairs, and the Actin11 S and Actin11 AS primer pairs were used to detect the expression of the SlJMJ524 gene by qRT-PCR to obtain PCR amplification products. The primer sequences are as follows:

[0062] Primer-qS: 5'-GCAGCAGTCGCTACCTAT-3' (SEQ ID NO. 6);

[0063] Primer-qAS: 5'-TGTCATTCCACCTACTCTTA-3' (SEQ ID NO. 7).

[0064] The conditions of qRT-PCR reaction were as follows: 94°C 10 min→[94°C 30 s→64°C 30 s]×40→59°C 30 s→72°C 10 min→94°C 2 min.

[0065] qRT-PCR used the comparative CT method (△△CT) to calculate gene expression, with the wild tomato SlActin11 gene as the internal reference gene and the untreated sample as the control. The difference in target gene expression was expressed by the multiple of the treated sample relative to the untreated sample at each time point. Each sample included 3 biological replicates and 3 technical replicates. The data was averaged from 3 biological replicates. If one value had a large deviation, the average of the two data was taken. The original data was standardized. The standardized data was analyzed for significant differences by T-test. Relative expression calculation method: 2 -△△CT =2 -(△CT处理-△CT对照) =2 -[(CT处理目的基因-CT处理内参基因)-(CT对照目的基因-CT对照内参基因)] The sequences of the internal reference gene primers are as follows:

[0066] Actin11 S: 5'-AAGATCCCATTCGTCCCCAT-3' (SEQ ID NO. 8);

[0067] Actin11 AS: 5'-CAAGAGCCTCAAGGAGAGTTGG-3' (SEQ ID NO. 9).

[0068] Test results such as Figure 1 As shown: Compared with the wild type, the expression levels of the overexpression lines SlJMJ524 were significantly increased. The lines with the highest expression levels of SlJMJ524, T2-generation SlJMJ524 overexpression line #2 and T2-generation SlJMJ524 overexpression line #8, were selected for the next phenotypic analysis.

[0069] Example 3. Obtaining the S1JMJ524 tomato gene knockout strain

[0070] 1. Obtain CRISPR / Cas9 vector: Use the website (http: / / crispr.hzau.edu.cn / CRISPR2 / ) to select two targets in the CDS region of SIJMJ524 gene and at the front end of the protein or in the important functional domain region, digest the two target sequences obtained with the pCBSG013 (NPTII) vector, and connect them. The obtained recombinant vector was sequenced and verified.

[0071] Target 1 (SEQ ID NO. 25): 5'-GATTTCTCCGGCCACGGCGTCGG-3';

[0072] Target 2 (SEQ ID NO. 26): 5′-CCGACGCCTTTCGAATCGCGAGG-3′;

[0073] The sequencing results showed that the recombinant vector pCBSG013(NPTII)-SlJMJ524 was a vector with the correct inserted fragment sequence and other sequences of the pCBSG013(NPTII) vector unchanged.

[0074] 2. Infect tomatoes (Micro-Tom) with Agrobacterium containing CRISPR / Cas9 vectors through Agrobacterium-mediated method. Take plump tomato (Micro-Tom) seeds and soak them at 55℃ for 15min, disinfect them with 75% ethanol for 30s, rinse them with sterile water 2-3 times, disinfect them with 2% sodium hypochlorite solution for 15min, rinse them with sterile water 6-7 times, dry them and inoculate them on 1 / 2MS culture medium, place them in the tissue culture room for dark culture, and then culture them under normal light culture conditions (16h light / 8h dark) when the seeds show white spots. After the cotyledons of the sterile seedlings are flattened, cut them into 0.5cm×0.5cm size and culture them on MS culture medium. Infect tomato cotyledons with Agrobacterium containing CRISPR / Cas9 vectors. Two days later, transfer the infected cotyledons to the screening medium containing 20mg / LG148 until the callus sprouts. When the adventitious buds grow to about 2-3 cm, they are cut off and inoculated into a rooting medium. After rooting, they are transferred to a nutrient pot containing vermiculite and peat soil in a ratio of 1:1 to obtain T0 generation transgenic SlJMJ524 gene knockout tomato seeds. After the surface of the tomato seeds is disinfected, they are sown on a 1 / 2MS medium containing 20 mg / L G148 to select and obtain T1 generation transgenic SlJMJ524 gene knockout tomato seedlings. This process is repeated until a T2 generation transgenic SlJMJ524 gene knockout tomato homozygous strain is obtained.

[0075] Extract genomic DNA from T2-generation SlJMJ524 knockout tomato seedlings and perform PCR identification. The specific steps are as follows:

[0076] Total DNA of T2-generation SlJMJ524 gene knockout tomato plants was extracted and used as a template. CRISPR Primer-S and CRISPR Primer-AS primers were used to knock out the target site of the plasmid by PCR to obtain PCR amplification products. The primer sequences are as follows:

[0077] CRISPR Primer-S: 5'-GGTGCCCTGAATGAACTCCA-3' (SEQ ID NO. 10);

[0078] CRISPR Primer-AS: 5'-AAAAGCGGCCATTTTCCACC-3' (SEQ ID NO. 11).

[0079] PCR amplification system (12.5 μl): cDNA 0.5 μl, CRISPR Primer-F 0.5 μl, CRISPR Primer-R 0.5 μl, Mix enzyme 6.25 μl, ddH2O 4.75 μl.

[0080] PCR amplification conditions:

[0081] SlJMJ524 knockout target PCR: 94°C for 2 min [94°C for 15 s → 58.3°C for 15 s → 72°C for 45 s] × 35 → 72°C for 10 min → 4°C to terminate the reaction;

[0082] The PCR amplification products were subjected to 1% agarose gel electrophoresis, and the detection results were as follows Figure 2 As shown: There is no amplification product in the PCR of wild-type tomato plants, but the target bands can be amplified in the T2-generation SlJMJ524 gene knockout tomato homozygous line #1 and the T2-generation SlJMJ524 gene knockout tomato homozygous line #2, indicating that the tomato gene SlJMJ524 has been knocked out and can be transcribed and expressed normally in transgenic tomatoes. The T2-generation SlJMJ524 gene knockout tomato homozygous line #1 and the T2-generation SlJMJ524 gene knockout tomato homozygous line #2 were selected for the next phenotypic analysis.

[0083] Example 4. Analysis of plant phenotype and physiological response indicators of SlJMJ524 transgenic tomato plants under cadmium stress

[0084] 1. Phenotype of transgenic SlJMJ524 tomatoes under cadmium treatment

[0085] Seeds of wild-type tomato WT, SlJMJ524 transgenic tomato T2 generation homozygous strains (#2, #8) and SlJMJ524 tomato gene knockout strains (#1, #2) were disinfected and cleaned, and then sown in nutrient pots containing vermiculite: peat soil 1:1. After the tomatoes grew for one week, 10 seedlings with excellent growth and uniform growth were selected from each strain and transferred to nutrient pots with CdCl2 concentration of 75μmol / L for stress treatment. The phenotype after stress was recorded by taking photos, and the root length and plant height were counted. All experiments were repeated 3 times for each technology and biological replicate. Ten strains were selected for each experiment.

[0086] The results are as follows Figure 3As shown in the figure: Under the control condition, the growth of each strain was almost the same. When the strain was stressed with 75μmol / L CdCl2, the plants were inhibited to a certain extent. However, the overexpression strains #2 and #8 were obviously more resistant than WT, with higher plant height and root length, while the knockout strains #1 and #2 were more sensitive, with shorter aboveground parts and taproots. This indicates that the SlJMJ524 gene is very likely to be involved in the process of plant response to cadmium stress, thereby improving the tolerance of plants to cadmium stress.

[0087] 2. Analysis of physiological indicators of transgenic SlJMJ524 tomato plants under cadmium stress

[0088] To further verify the tolerance of transgenic SlJMJ524 tomatoes to cadmium stress, the antioxidant enzyme activities, the content of related pigments in the photosynthetic system and the active oxygen scavenging ability of transgenic SlJMJ524 tomatoes in the seedling stage were measured after being treated with 75 μmol / L CdCl2 for 0 h, 36 h and 72 h. Figure 4As shown: At 0h of stress, all indicators of tomato strains showed no significant differences, indicating that the strains were less damaged when not under stress. At 36h of Cd stress, the POD, SOD, and CAT contents of all strains increased. Compared with the WT strain in the same group, the POD, SOD, and CAT contents of the SlJMJ524 overexpression strain were higher, while the POD, SOD, and CAT contents of the SlJMJ524 knockout strain were lower than those of the WT strain. At 72h of Cd stress, the POD, SOD, CAT, and MDA contents of all strains increased. Compared with the WT strain in the same group, the POD, SOD, and CAT contents of the SlJMJ524 overexpression strain were higher and basically reached a significant level, while the POD, SOD, and CAT contents of the SlJMJ524 knockout strain were significantly lower than those of the WT strain. MDA content represents the degree of damage to plants under stress. At 36h of Cd stress, the MDA content of each strain increased, but the MDA content of SlJMJ524 overexpression strain was significantly lower than that of WT and SlJMJ524 knockout strains. At 72h of Cd stress, each strain showed the same situation as at 36h, proving that the damage to SlJMJ524 overexpression strain was lighter than that to the other two strains. At 36h and 72h of Cd stress, the chlorophyll a, chlorophyll b, carotenoids and total chlorophyll contents of each strain were slightly lower than those of the control group, but the overall trend was consistent. Compared with the WT strain in the same group, the contents of various photosynthetic pigments in the SlJMJ524 overexpression strain were significantly increased, while the contents of the SlJMJ524 gene knockout strain were significantly lower than those in the same group of WT, indicating that SlJMJ524 overexpression can improve the photosynthetic capacity of plants under cadmium stress and thus make plants resistant to cadmium. The leaves of each strain were stained with NBT and DAB. Under normal conditions, the staining of each strain was lighter, indicating less accumulation of superoxide anions. After cadmium stress, it was clearly found that the SlJMJ524 overexpression strain had fewer brown and blue spots than the WT. On the contrary, the SlJMJ524 knockout strain had the most brown and blue spots on its leaves, indicating that its superoxide anion content accumulated the most. The determination of its hydrogen peroxide content and superoxide anion content also further proved that the SlJMJ524 overexpression strain was more resistant to cadmium. In summary, the overexpression of the SlJMJ524 gene can make tomatoes resistant to Cd by enhancing the activity of plant antioxidant enzymes, photosynthetic capacity, and reactive oxygen scavenging capacity.

[0089] Example 5. Determination of the expression level of marker genes related to cadmium stress in transgenic tomatoes

[0090] 1. Processing of plant materials

[0091] The seeds of wild-type tomato WT, SlJMJ524 transgenic tomato T2 generation homozygous strains (#2, #8) and SlJMJ524 tomato gene knockout strains (#1, #2) were disinfected and cleaned, and then sown in nutrient pots containing vermiculite: peat soil in a ratio of 1:1. After the tomatoes grew for one week, the seedlings with excellent growth and uniform growth were selected from each strain and transferred to nutrient pots with a CdCl2 concentration of 75 μmol / L for stress treatment. The plant materials SlJMJ524 transgenic overexpression tomato strains #2, #8, SlJMJ524 gene knockout tomato strains #1, #2 and wild-type WT obtained after the treatment were cut as tissue samples to be tested, quickly frozen in liquid nitrogen, and then stored at -80°C.

[0092] 2. Extraction of total RNA and acquisition of cDNA

[0093] The total RNA of the tissue samples to be tested after stress and the tissue samples of the different strains without stress obtained in the above step 1 were respectively extracted using the Plant Total RNA Isolation Kit (Foregene); the total RNA was used as a template for reverse transcription to obtain cDNA.

[0094] 3. Real-time PCR

[0095] Using the above cDNA as a template and cadmium stress-related marker gene primers, the expression level of the SlJMJ524 gene was detected by Real-time PCR. The primer sequences are as follows:

[0096] Cadmium stress-related marker genes:

[0097] APX1-S: 5'-CTGATGTTCCCTTTCACCCTG-3' (SEQ ID NO. 12);

[0098] APX1-AS: 5'-ATTTCAATAGAAGTTCCCAGTAGCA-3' (SEQ ID NO. 13).

[0099] APX2-S: 5'-TCAGTGATCCTGCTTTCCGC-3' (SEQ ID NO. 14);

[0100] APX2-AS: 5'-TGTCACCACCCTCCCAACTCT-3' (SEQ ID NO. 15).

[0101] POD-S: 5'-CTTGCCCTAATGCTCTCACC-3' (SEQ ID NO. 16);

[0102] POD-AS: 5'-GCATCACAACCCTGAACAAA-3' (SEQ ID NO. 17).

[0103] P5CS-S: 5'-TGCTGTAGGTGTTGGTCGTCA-3' (SEQ ID NO. 18);

[0104] P5CS-AS: 5'-TGCCATCAAGCTCAGTTTGTG-3' (SEQ ID NO. 19).

[0105] GSH1-S: 5'-CTGCATTCTGGGTGGGT-3' (SEQ ID NO. 20);

[0106] GSH1-AS: 5'-CTCGGCTACTTCGTTCA-3' (SEQ ID NO. 21).

[0107] NRAMP3-S: 5'-TGGTTAACTGGATTGTTGGCTGCTGGAC-3' (SEQ ID NO. 22);

[0108] NRAMP3-AS: 5'-ATGAATTCAAAGAAGGGGGATCAGGGCA-3' (SEQ ID NO. 23).

[0109] Real-time PCR reaction conditions: 94°C 10 min→[94°C 30 s→64°C 30 s]×40→59°C 30 s→72°C 10 min→94°C 2 min.

[0110] Real-time PCR uses the comparative CT method (△△CT method) to calculate gene expression, with the wild tomato SlActin11 gene as the internal reference gene and the untreated sample as the control. The difference in target gene expression is expressed by the multiple of the treated sample relative to the untreated sample at each time point. Each sample includes 3 biological replicates and 3 technical replicates. The data is the average of the 3 biological replicates. If one value has a large deviation, the average of the two data is taken. The original data is standardized. The standardized data is analyzed for significant differences by T-test. Relative expression calculation method: 2 -△△CT =2 -(△CT处理-△CT对照) =2 -[(CT处理目的基因-CT处理内参基因)-(CT对照目的基因-CT对照内参基因)] The sequences of the internal reference gene primers are as follows:

[0111] Actin11-S: 5'-AAGATCCCATTCGTCCCCAT-3';

[0112] Actin11-AS: 5'-CAAGAGCCTCAAGGAGAGTTGG-3'.

[0113] The results are as follows Figure 5 As shown: After treatment with 75 μmol / L CdCl2, the relative expression levels of 6 cadmium stress-related marker genes in WT and SlJMJ524 transgenic tomatoes were analyzed. After stress treatment, the relative expression levels of the 6 genes in transgenic tomatoes increased except GSH1, and the difference was extremely significant compared with WT. Among them, the gene expression levels of APX1, APX2, and POD related to antioxidant enzymes in the five strains increased under Cd stress, and the expression level of antioxidant enzyme-related genes in the SlJMJ524 overexpression strain was the highest, and the expression level of antioxidant enzyme-related genes in the SlJMJ524 gene knockout strain was the lowest. The genes GSH1 and P5CS related to the plant chelation pathway, among which the gene expression levels of P5CS in the five strains increased under Cd stress, and the expression level of antioxidant enzyme-related genes in the SlJMJ524 overexpression strain was the highest, and the expression level of antioxidant enzyme-related genes in the SlJMJ524 knockout strain was the lowest; the expression level of GSH1 gene in the five strains after Cd stress was slightly lower than that in the control group, but the expression level of GSH1 in the SlJMJ524 overexpression strain was still significantly higher than that in other strains after stress. NRAMP3 is a metal transporter-related gene. The gene expression levels of the five strains under Cd stress were consistent with the expression levels of the other three types of genes. The NRAMP3 gene was expressed at the highest level in the SlJMJ524 overexpression strain and the lowest level in the SlJMJ524 knockout strain, and all of them reached significant levels. The above results showed that under cadmium stress conditions, stress marker genes such as APX1, APX2, and POD were upregulated in SlJMJ524 overexpressing transgenic tomatoes, and the expression levels were significantly higher than those in WT. Therefore, the SlJMJ524 gene may enhance tolerance to cadmium stress by regulating the expression levels of stress-induced marker genes.

Claims

1. Application of SlJMJ524 protein in improving cadmium resistance of tomatoes, characterized in that: The amino acid sequence of the SlJMJ524 protein is shown in SEQ ID NO.

24.

2. Application of SlJMJ524 gene in improving cadmium resistance of tomatoes, characterized in that: The nucleic acid molecule of the SlJMJ524 gene is shown in SEQ ID NO.

3.

3. Application of a recombinant vector containing the SlJMJ524 gene in improving the cadmium resistance of tomatoes, characterized in that: The nucleic acid molecule of the SlJMJ524 gene is shown in SEQ ID NO.

3.

4. The use according to claim 3, characterized in that: The starting vector of the recombinant vector is pCAMBIA2300.

5. The use of recombinant microbial cells containing the SlJMJ524 gene in improving the cadmium resistance of tomatoes, characterized in that: The nucleic acid molecule of the SlJMJ524 gene is shown in SEQ ID NO.

3.

6. The use according to claim 5, characterized in that: The recombinant microbial cell is a eukaryotic microbial cell or a prokaryotic microbial cell.

7. Use of tomatoes overexpressing the SlJMJ524 gene in improving cadmium resistance, characterized in that: The nucleic acid molecule of the SlJMJ524 gene is shown in SEQ ID NO.

3.

8. A method for improving cadmium resistance of tomatoes, characterized in that: The specific steps of the method are as follows: Step 1: Connect the SlJMJ524 gene to the pCAMBIA2300 vector to obtain a recombinant vector; Step 2: Transform the recombinant vector obtained in step 1 into Agrobacterium to obtain recombinant Agrobacterium; Step 3: Infect tomatoes with the recombinant Agrobacterium obtained in step 2 to obtain transgenic tomatoes.

9. The method according to claim 8, characterized in that In step 1, the primers for amplifying the SlJMJ524 gene are SEQ ID NO.1 and SEQ ID NO.

2.

10. The method according to claim 8, characterized in that The nucleic acid molecule of the SlJMJ524 gene in step 1 is shown in SEQ ID NO.3.

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