Application of cadmium resistance gene in improving cadmium resistance of tomato

Abstract

The application discloses application of an anti-cadmium gene in improving cadmium resistance of tomatoes and belongs to the technical field of plant stress resistance. The application aims to provide a method for improving cadmium resistance of tomatoes. The application discloses application of a protein with an amino acid sequence as shown in SEQ ID NO. 28 in improving cadmium resistance of tomatoes. The SlJMJ gene can enhance the tolerance to cadmium stress by regulating the expression level of a stress-induced marker gene.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant stress resistance, and particularly relates to application of a cadmium-resistant gene in improving cadmium resistance of tomatoes. BACKGROUND

[0002] Cadmium (Cd) is one of the most harmful and widely distributed toxic heavy metal pollutants in agricultural soil, and has strong biological persistence. It exists for a long time after being consumed by organisms. According to the investigation and evaluation of the Ministry of Ecology and Environment, the heavy metal pollution of cadmium in cultivated soil mainly comes from non-ferrous metal smelting, mining and other industries, and enters the soil through water transport or atmospheric deposition; the pollutants on the land of industrial and mining enterprises mainly come from chemical, petrochemical and coking industries, and the toxic and harmful substances enter the soil through leakage, loss and dispersion. Due to rapid industrialization and urbanization, there are great challenges in protecting cultivated soil from heavy metal pollution. The use of cereals, vegetables and root crops planted in contaminated soil raises concerns about food safety and human health. In terms of pollutant over-standard conditions, the point over-standard rate of cadmium is 7.0%, ranking first among common inorganic pollutants.

[0003] Tomato is one of the vegetables with a large amount of consumption in human daily life, and the quality thereof directly affects the daily food safety of people. Tomato is one of the vegetables that can easily absorb and accumulate cadmium. Under the stress of 2.0 mg / kg Cd, the over-standard rate of cadmium in the edible parts of all tested different varieties of tomatoes is 100%, and the excessive accumulation of cadmium in tomatoes can pose a great threat to human health. Therefore, it is a technical problem to be solved to find a method for cadmium stress tolerance of tomatoes. SUMMARY

[0004] The application aims to provide a method for cadmium stress tolerance of tomatoes.

[0005] The application provides application of a protein with an amino acid sequence as shown in SEQ ID NO. 28 in improving cadmium resistance of tomatoes.

[0006] The application provides application of a gene with a nucleic acid sequence as shown in SEQ ID NO. 3 in improving cadmium resistance of tomatoes.

[0007] The application provides application of a recombinant vector containing a gene with a nucleic acid sequence as shown in SEQ ID NO. 3 in improving cadmium resistance of tomatoes.

[0008] Further, the starting vector of the recombinant vector is pCAMBIA2300.

[0009] The application provides application of a recombinant microbial cell containing a gene with a nucleic acid sequence as shown in SEQ ID NO. 3 in improving cadmium resistance of tomatoes.

[0010] Further limit, the recombinant microbial cell is a eukaryotic microbial cell or a prokaryotic microbial cell.

[0011] The application provides application of a tomato plant overexpressing a nucleic acid molecule, such as a gene shown in SEQ ID NO. 3, in improving cadmium resistance.

[0012] The application provides a method for improving cadmium resistance of a tomato, and specific steps of the method are as follows.

[0013] Step 1: connecting a gene shown in SEQ ID NO. 3 with a vector to obtain a recombinant vector;

[0014] Step 2: transferring the recombinant vector obtained in step 1 into Agrobacterium to obtain a recombinant Agrobacterium;

[0015] Step 3: infecting a tomato with the recombinant Agrobacterium obtained in step 2 to obtain a transgenic tomato.

[0016] Further limit, the primer for amplifying the gene shown in SEQ ID NO. 3 in step 1 is SEQ ID NO. 1 and SEQ ID NO. 2.

[0017] Further limit, the vector in step 1 is pCAMBIA2300.

[0018] Beneficial effects: a tomato cadmium tolerance gene sequence is shown as SEQ ID NO. 3, and an overexpression vector and a mutant thereof are constructed, and through Agrobacterium tumefaciens-mediated tomato homologous transformation method, the overexpression tomato of the gene shown as SEQ ID NO. 3 and the sljmj mutant tomato are successfully obtained. The experiment of the application proves that overexpression of the gene in tomato can enhance the tolerance of tomato seedling stage to cadmium stress, and the sljmj mutant shows lower tolerance to cadmium stress. It is proved that the gene can lay a foundation for cultivating transgenic plants with cadmium tolerance. After cadmium stress treatment, the plant phenotype, dry weight and fresh weight and related physiological indexes are analyzed, and the results show that compared with the control, the plant phenotype of SlJMJ-OE tomato is better, the root length, plant height and fresh weight and other values are larger, the plant phenotype of sljmj (jmj) mutant tomato is the worst, and the root length, plant height and fresh weight and other values are smaller; the H2O2 content of SlJMJ-OE (JMJ-OE) is lower, and the antioxidant enzyme activities such as SOD, POD, CAT and APX are higher, and the sljmj mutant is opposite. Such research results confirm that the SlJMJ gene can enhance the resistance of tomato to cadmium stress. The expression of stress-related marker genes of transgenic tomato is analyzed, and it is found that under the condition of cadmium stress, the stress marker genes such as SlCAT1, SlPOD and SlSOD are up-regulated in SlJMJ-OE transgenic tomato, and the expression amount is significantly higher than that of WT, and are down-regulated in sljmj mutant tomato, and the expression amount is significantly lower than that of WT. Therefore, the SlJMJ gene can enhance the tolerance to cadmium stress by regulating the expression level of stress-induced marker genes. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 PCR amplification of SlJMJ gene;

[0020] Figure 2 PCR detection diagram of SlJMJ overexpression tomato positive seedlings;

[0021] Figure 3 SlJMJ overexpression tomato RNA level identification diagram;

[0022] Figure 4 SlJMJ mutant tomato positive seedling identification diagram;

[0023] Figure 5 SlJMJ overexpression and mutant tomato phenotype, plant height and root length under cadmium stress;

[0024] Figure 6 Photosynthetic pigment content of SlJMJ transgenic tomato under cadmium stress;

[0025] Figure 7 Antioxidant enzyme activity of SlJMJ transgenic tomato under cadmium stress;

[0026] Figure 8 ROS and MDA of SlJMJ transgenic tomato under cadmium stress;

[0027] Figure 9 Expression analysis of Marker genes related to cadmium stress. DETAILED DESCRIPTION

[0028] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0029] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0030] The DH5α competence and Agrobacterium GV3101 competence and Agrobacterium K599 competence used in the following examples are products of Invitrogen Corporation.

[0031] The Micro-Tom tomato used in the following examples can be obtained from commercial channels.

[0032] Example 1. Cloning of tomato SlJMJ gene

[0033] 1. Treatment of plant material

[0034] Full and ripe Micro-Tom tomato seeds were washed with sterile water for 3-4 times and then placed on a wet filter paper, and germinated at 25°C in the dark for 4 days. When the sprouts grew to about 1-2 cm, they were transferred to pots containing nutrient soil (peat soil: vermiculite = 2:1) and placed in a greenhouse for cultivation. The growth conditions of the greenhouse were 25°C / 20°C (day / night), and the light cycle was 16 hours light / 8 hours dark. When the seedlings grew to 4 leaves 1 heart, their leaves were taken into EP tubes and stored at -80°C.

[0035] 2. RNA extraction

[0036] The total RNA of the leaves of the above tomato seedlings was extracted using Plant Total RNA Isolation Kit (Foregene).

[0037] 3. Obtaining of cDNA

[0038] The above total RNA was used as a template for reverse transcription to obtain cDNA.

[0039] 4. PCR amplification

[0040] The above cDNA was used as a template for PCR amplification using Primer-KS and Primer-KAS primers to obtain PCR amplification products. The primer sequences are as follows:

[0041] Primer-KS: 5'-CGGGGTACCATTAGGTTTTTGAATTGTGATGGAT-3' (SEQ ID NO. 1);

[0042] Primer-KAS: 5'-AACTGCAGGCAGTCATTCTTTTGTAAATTTCAC-3' (SEQ ID NO. 2).

[0043] PCR amplification system (50 μl): cDNA 1 μl, Primer-KS 1.5 μl, Primer-KAS 1.5 μl, KOD plus enzyme 1 μl, ddH2O 32 μl, 10 x PCR Buffer Mix 5 μl, 2 mM dNTP 5 μl, MgSO4 3 μl.

[0044] PCR amplification conditions: 94°C for 2 min, [98°C for 10 s, 54°C for 30 s, 68°C for 1 min] for 35 cycles; 68°C for 5 min; 4°C to terminate the reaction.

[0045] The PCR amplification product was detected by 1% agarose gel electrophoresis, as shown in Figure 1 , a band with a molecular weight of about 1041 bp was obtained, and the PCR amplification product was recovered by agarose gel extraction kit (Omega Gel Extraction kit); it was connected with pCAMBIA-2300 vector to obtain a recombinant plasmid, which was named pCAMBIA2300-SlJMJ, and then it was transformed into E. coli DH5α competent cells and sent for sequencing.

[0046] The sequencing results showed that the PCR amplification product with a size of 1041 bp was obtained, which was named SlJMJ gene, and its nucleotide sequence was shown in SEQ ID NO. 3.

[0047] SlJMJ gene sequence: (SEQ ID NO. 3)

[0048] ATGGATGATATACCTGAATGGCTTAAAGGGTTGCCCCTCGCACCTGAATTTCGACCTACT

[0049] GATACTGAATTTGCTGACCCCATTGCCTATATATCAAAAATCGAGAAGGAAGCTAGTGCT

[0050] TTTGGTATATGTAAGGTCATTCCGCCATTGCCTAAACCTTCAAAGAAGTATGTCCTTCAT

[0051] AACTTGAACAACTCTCTATCAAAGTGCCCTGACCTCAATTCTGCTGGTGCCCCTGTTTTT

[0052] ACCACCCGACATCAGGAATTGGGTCATACTGAAAAGAAGAAATTCCCATTTGGAGCAC

[0053] AGAAGCAAGTCTGGCAAAGTGGACAACTTTATACGCTAGACCAGTTCGAAACCAAGTC

[0054] CAAGAACTTTGCCAGGACTCAATTCGGCATCGTTAAGGACATTTCTCCTTTCCTTGTCG

[0055] AGGCAATGTTTTGGAAAACCGCTTTTGACCACCCTATCTACGTCGAGTATGCAAATGAC

[0056] GTGCCTGGTTCTGCCTTTGGGGAGCCAGAGGAGAATTTTTGCAGGACAAAAAGACCAC

[0057] GTAACAGGAAAATTCTTGATAGAACAAGTTCGACTACTAGTGTGGACAAAGGTCGGTC

[0058] TCATCATAGTGTAGACACACCATCCTCTTCTTTGTTAACCCCTTTATCAAACTCATCCCCT

[0059] TTTAGGCCAAAAGGTTGTAGTAATGCCGCTGAAATGGAAGGTAGTGCTGGTTGGAAGC

[0060] TTGCAAACAGTCCTTGGAATTTGCAAGTAATTGCGCGGTCCCCTGGGTCACTCACTCGC

[0061] TTCATGCCTGATGATATCCCTGGTGTTACTTCTCCAATGGTCTATATAGGGATGCTGTTTA

[0062] GCTGGTTTGCTTGGCATGTTGAGGATCATGAGCTTCACAGTCTAAATTTTCTTCATACAG

[0063] GATCTCCTAAGACTTGGTATGCAGTGCCAGGAGATTATGCATTTAGTTTTGAAGAAGTCA

[0064] TTCGTTGTCATGCTTATGGAGAAACTACTGATCGATTAGGTATGAATACATACTTTTTTGTCAACAACAATGTGAAATTTACAAAAGAATGA;

[0065] SlJMJ amino acid sequence: (SEQ ID NO. 28)

[0066] MDDIPEWLKGLPLAPEFRPTDTEFADPIAYISKIEKEASAFGICKVIPPLPKPSKKYVLHNLN

[0067] NSLSKCPDLNSAGAPVFTTRHQELGHTEKKKFPFGAQKQVWQSGQLYTLDQFETKSKNFA

[0068] RTQFGIVKDISPFLVEAMFWKTAFDHPIYVEYANDVPGSAFGEPEENFCRTKRPRNRKILDR

[0069] TSSTTSVDKGRSHHSVDTPSSSLLTPLSNSSPFRPKGCSNAAEMEGSAGWKLANSPWNLQV

[0070] IARSPGSLTRFMPDDIPGVTSPMVYIGMLFSWFAWHVEDHELHSLNFLHTGSPKTWYAVPGDYAFSFEEVIRCHAYGETTDRLGMNTYFFVNNNVKFTKE.

[0071] Example 2. Obtaining of SlJMJ-OE plants

[0072] I. Obtaining of SlJMJ overexpression recombinant plasmid

[0073] 1. Obtaining of SlJMJ gene

[0074] The pCAMBIA2300-SIJMJ obtained in step 4 in Example 1 was used as a template, and primer Primer-ES and Primer-EAS were used for PCR amplification to obtain a PCR amplification product, i.e. the SlJMJ gene. The primer sequences are as follows:

[0075] Primer-ES: 5'-CGGGGTACCATTAGGTTTTTGAATTGTGATGGAT-3' (SEQ ID NO. 4);

[0076] Primer-EAS: 5'-AACTGCAGGCAGTCATTCTTTTGTAAATTTCAC-3' (SEQ ID NO. 5).

[0077] The PCR amplification system (25 μl) was as follows: cDNA 1 μl, Primer-F 1 μl, Primer-R 1 μl, Prime Star Mix 12.5 μl, and ddH2O 9.5 μl.

[0078] The PCR amplification conditions were as follows: 98℃ 10 s, 58℃ 10 s, 72℃ 1 min 30 s, 35 cycles; 72℃ 5 min; and 4℃ termination.

[0079] 2. Obtaining of a plant expression vector

[0080] The pCAMBIA 2300 vector and the above-mentioned PCR amplification product were subjected to restriction enzyme digestion, ligation, to obtain a recombinant vector pCAMBIA 2300-SIJMJ. And it was subjected to sequencing verification.

[0081] The sequencing result shows that the recombinant vector pCAMBIA 2300-SIJMJ is a vector obtained by replacing the DNA fragment between the enzyme digestion sites of the pCAMBIA 2300 vector with the SlJMJ gene shown in sequence 1 in the sequence listing, and keeping other sequences of the pCAMBIA 2300 vector unchanged.

[0082] 3. Transformation

[0083] The recombinant vector pCAMBIA 2300-SIJMJ was transformed into Agrobacterium tumefaciens GV3101 by heat shock method, and positive transformants were obtained by PCR identification, which were used for infestation of tomato plants.

[0084] II. Obtaining of SlJMJ overexpression and mutant tomato plants

[0085] 1. Seed washing

[0086] Select full tomato seeds, 55℃ soak 15min, 75% ethanol disinfection 30s, rinse with sterile water 2-3 times, 2min each time, 2% sodium hypochlorite solution disinfection 15min, rinse with sterile water 6-7 times, 2min each time.

[0087] 2, inoculation

[0088] In the clean bench, the sterilized seeds are placed on the sterilized filter paper, the water is absorbed, the seeds are inoculated into the sterilized 1 / 2MS solid medium, the tissue culture bottle is placed in the tissue culture room for dark culture, and when the seed white point appears, it is then placed in the normal light culture condition (16h light / 8h dark) for culture.

[0089] 1 / 2MS solid medium: 2.37g / L MS+30g / L sucrose+7g / L agar

[0090] 3, pre-culture

[0091] The cotyledon of the aseptic seedling is flattened and the seedling is cut. The aseptic seedling is clamped to the glass culture dish with filter paper, the cotyledon is cut into about 0.5cm×0.5cm size, the cotyledon back is placed downward, and the pre-culture medium is placed in the normal light culture condition for 2 days.

[0092] Pre-culture medium: 4.74g / L MS+30g / L sucrose+7g / L agar+2mg / L ZT+0.1mg / L IAA

[0093] 4, co-culture

[0094] Prepare sterilized beaker, filter screen, filter paper, glass culture dish and tweezers in advance.

[0095] Start preparing Agrobacterium: take 1mL of preserved glycerol bacteria and transfer to 100mL LB liquid medium with Rif and Kan, 28℃, 200rpm overnight culture, centrifuge at 5000rpm for 5min, pour off the supernatant after centrifugation, add liquid MS to resuspend, OD is about 0.6 (diluted bacterial solution), 50mL bacterial solution+50mL MS.

[0096] Gently clamp the pre-cultured explant with tweezers and put it into the beaker, pour in the bacterial solution, contact with the bacterial solution for 10min, then put the explant back into the pre-culture medium, dark culture for 2d, cotyledon back upward.

[0097] 5, screening

[0098] After co-culture, the cotyledon is transferred to the selection medium, the cotyledon back is downward. When the cotyledon swells and green bud points grow, it is transferred to new medium for continuous screening until the callus sprouts.

[0099] Screening medium: 4.74 g / L MS + 30 g / L sucrose + 7 g / L agar + 2 mg / L ZT + 0.1 mg / L IAA + 300 mg / L Tim + 50 mg / L Kan

[0100] 6. Rooting

[0101] After the adventitious buds grow, the callus is cut off, and 2-3 cm of the bud is cut off and connected to the rooting medium.

[0102] Rooting medium: 1 / 2MS + 0.5 mg / L IAA + 50 mg / L Kan + 300 mg / L Tim

[0103] 7. Domestication

[0104] Selecting the root system, the seedling height is 4-6 cm, and the growth is vigorous. The seedlings are washed with the culture medium, and the culture medium is mixed with 2:1 ratio of peat soil and vermiculite. The washed root seedlings are transplanted in the pot. After transplanting, the film is opened, and the normal management is carried out.

[0105] III. Identification of overexpression positive lines

[0106] 1. Identification of SlJMJ overexpression tomato positive seedlings

[0107] Take 0.1 g of the leaf of the T0 generation SlJMJ overexpression seedling, extract DNA, and perform PCR identification. The specific steps are as follows:

[0108] Extract the DNA of the T0 generation SlJMJ overexpression plant obtained after domestication, and use the sequence of the 35S promoter connected to the target gene SlJMJ to design primers and perform PCR detection. The primer sequence is as follows:

[0109] 35S / J12-F: 5'-ACTGTCCTTTTGATGAAGTGACAGATAGC-3'(SEQ ID NO. 6);

[0110] 35S / J12-R: 5'-AACAAAGAAGAGGATGGTGTGTCTAC-3'(SEQ ID NO. 7).

[0111] PCR amplification system (25 μl): cDNA 1 μl, Primer-F 1 μl, Primer-R 1 μl, Mix 12.5 μl, ddH2O 9.5 μl.

[0112] PCR amplification conditions: 94℃ 2min→ [94℃ 15s→ 58℃ 30s→ 72℃ 1min 30s] x 35 cycles→ 72℃ 10min→ 4℃ stop reaction;

[0113] The PCR amplification product was subjected to 1% agarose gel electrophoresis, and the detection results are shown in Figure 2 As shown in the table, the transgenic plants can amplify a specific band of 1042bp, while the wild type plants do not amplify a specific band. Among them, 18 resistant seedlings were positive in PCR detection.

[0114] 2. Identification of SlJMJ overexpression tomato RNA level

[0115] PCR detection of positive tomato seedlings was carried out by fluorescent quantitative PCR. The specific steps are as follows:

[0116] PCR detection of positive tomato seedlings was carried out by fluorescent quantitative PCR. The specific steps are as follows:

[0117] SlJMJ-qF: 5'-ACCGCTTTTGACCACCCTATC-3'(SEQ ID NO. 8);

[0118] SlJMJ-qR: 5'-TTCCAACCAGCACTACCTTCC-3'(SEQ ID NO. 9).

[0119] PCR amplification conditions: 95℃ 10min→ [95℃ 30s→ 58℃ 30s→ 72℃ 45s] x 40→ 72℃ 10min→ 95℃ 30s

[0120] The results are shown in Figure 3 As shown in the table, the transgenic plants can amplify a specific band of 1042bp, while the wild type plants do not amplify a specific band. Among them, 18 resistant seedlings were positive in PCR detection.

[0121] Example 3. Obtaining of SlJMJ mutant plants

[0122] I. Obtaining of SlJMJ mutant recombinant plasmid

[0123] 1. Using the CRISPR-P website of HZAU (http: / / crispr.hzau.edu.cn / CRISPR2 / ) to select two target sites in the CDS region of the SlJMJ gene and as far as possible in the front of the protein or the important functional domain region, the target site sequences are as follows: Target 1: 5'-ACCCAATTCCTGATGTCGGGTGG-3' (SEQ ID NO. 10); Target 2: 5'-CATACTCGACGTAGATAGGGTGG-3' (SEQ ID NO. 11), both of which are connected to the CRISPR / Cas9 knockout vector. After designing, the SlJMJ gene CRISPR / Cas9 knockout vector is constructed by Jiangsu Wemi Biotechnology Co., Ltd.

[0124] 2. Transformation

[0125] The mutant plasmid is transformed into Agrobacterium tumefaciens GV3101 by heat shock method, and positive transformants are obtained by PCR identification, which are used to infect tomato plants. The PCR primers are as follows:

[0126] Cas9-F: 5'-GGTGCCCTGAATGAACTCCA -3' (SEQ ID NO. 12);

[0127] Cas9-R: 5'-AAAAGCGGCCATTTTCCACC-3' (SEQ ID NO. 13).

[0128] PCR amplification system (25 μl): cDNA 1 μl, Cas9-F 1 μl, Cas9-R 1 μl, Mix 12.5 μl, ddH2O 9.5 μl.

[0129] PCR amplification conditions: 94℃ 2min, [94℃ 30s, 58℃ 30s, 72℃ 30s] x 35 cycles; 72℃ 10min; 4℃ termination reaction.

[0130] II. Obtaining of SlJMJ overexpression and mutant tomato plants

[0131] 1. Seed washing

[0132] Select full tomato seeds, soak at 55℃ for 15min, sterilize with 75% ethanol for 30s, rinse with sterile water for 2-3 times, each time for 2min, sterilize with 2% sodium hypochlorite solution for 15min, rinse with sterile water for 6-7 times, each time for 2min.

[0133] 2. Inoculation

[0134] In a clean bench, sterilized seeds were placed on sterilized filter paper, and water was absorbed. The seeds were inoculated on sterilized 1 / 2MS solid medium, and the tissue culture bottles were placed in a tissue culture room for dark culture. When the seeds showed white spots, the seeds were placed in normal light culture conditions (16h light / 8h dark) for culture.

[0135] 1 / 2MS solid medium: 2.37g / L MS+30g / L sucrose+7g / L agar

[0136] 3. Pre-culture

[0137] The cotyledon of the aseptic seedling was flattened and the seedling was cut. The aseptic seedling was picked up with tweezers and placed on a glass culture dish with filter paper. The cotyledon was cut into about 0.5cm x 0.5cm, and the back of the cotyledon was placed on the pre-culture medium and cultured under normal light culture conditions for 2 days.

[0138] Pre-culture medium: 4.74g / L MS+30g / L sucrose+7g / L agar+2mg / L ZT+0.1mg / L IAA

[0139] 4. Co-culture

[0140] Sterilized beakers, filter paper, glass culture dishes, and tweezers were prepared in advance.

[0141] The preparation of Agrobacterium was started. 1mL of preserved glycerol bacteria was transferred to 100mL of LB liquid medium containing Rif and Kan, and cultured overnight at 28°C and 200rpm. After centrifugation at 5000rpm for 5min, the supernatant was discarded, and liquid MS was added for resuspension. The OD was about 0.6 (after dilution), and 50mL of bacterial solution was added to 50mL of MS.

[0142] The pre-cultured explants were gently picked up with tweezers and placed in a beaker. After 10min of contact with the bacterial solution, the explants were placed back in the pre-culture medium and cultured in the dark for 2d, with the back of the cotyledon facing up.

[0143] 5. Screening

[0144] After co-culture, the cotyledon was transferred to the selection medium, with the back of the cotyledon facing down. When the cotyledon swelled and green sprout points appeared, it was transferred to a new medium for continued screening until the callus sprouted.

[0145] Screening medium: 4.74g / L MS+30g / L sucrose+7g / L agar+2mg / L ZT+0.1mg / L IAA+300mg / L Tim+50mg / L Kan

[0146] 6. Rooting

[0147] When the adventitious buds grow, the callus is cut off, and the 2-3 cm buds are cut and inoculated into the rooting medium.

[0148] Rooting medium: 1 / 2MS + 0.5 mg / L IAA + 50 mg / L Kan + 300 mg / L timentin

[0149] 7. Domestication

[0150] The tissue culture seedlings with thick roots, 4-6 cm high, and vigorous growth are selected, and the seedlings are semi-opened for 2 days. The tissue culture seedlings are taken out of the culture medium, and the residual culture medium on the roots is washed. The peat soil and vermiculite are mixed in a ratio of 2:1, placed in a flowerpot, and watered to saturation. The washed rooting seedlings are transplanted therein. After transplantation, the film is covered and the humidity is maintained, and the flowerpot is placed in a greenhouse at 25°C. After 5-7 days, the film is opened, and normal management is carried out.

[0151] III. Identification of positive tomato mutant seedlings

[0152] 0.1 g of leaf of T0 generation SlJMJ mutant seedlings is taken, DNA is extracted, and PCR identification is carried out. The specific steps are as follows:

[0153] The DNA of T0 generation SlJMJ mutant plants obtained after domestication is extracted, and PCR detection is carried out using Cas9-F / R primers with the DNA as a template. The primer sequences are as follows:

[0154] Cas9-F: 5'-GGTGCCCTGAATGAACTCCA-3';

[0155] Cas9-R: 5'-AAAAGCGGCCATTTTCCACC-3'.

[0156] PCR amplification system (25 μl): cDNA 1 μl, Cas9-F 1 μl, Cas9-R 1 μl, Mix 12.5 μl, ddH2O 9.5 μl.

[0157] PCR amplification conditions: 94°C for 2 min, [94°C for 30 s, 58°C for 30 s, 72°C for 30 s] x 35 cycles; 72°C for 10 min; 4°C for termination reaction.

[0158] The PCR amplification product is subjected to 1% agarose gel electrophoresis, and the detection result is as shown in Figure 4 : The mutant plants can amplify a specific band of 478 bp, and the wild type plants do not amplify a specific band. Among them, 28 strains of jmj mutant plants are positive in PCR detection.

[0159] Example 4. Functional verification of SlJMJ overexpression and mutant plants under cadmium stress

[0160] I. Determination of seedling growth index of overexpression and mutant under cadmium treatment

[0161] The seeds of wild type tomato WT, T2 generation homozygous line OE13 and OE15 of SlJMJ overexpression, and T2 generation homozygous line jmj12-4 and jmj12-10 of mutant were washed with sterile water for 3-4 times, then placed on wet filter paper, and germinated in dark at 25℃ for 4 days. When the buds grew to about 1-2 cm, they were transferred to pots containing nutrient soil (peat soil: vermiculite = 2:1), and placed in a greenhouse for culture. The growth conditions of the greenhouse were 25℃ / 20℃ (day / night), and the light cycle was 16 hours light / 8 hours dark. After the seedlings grew to four leaves, they were transferred to water solution for stress treatment (CdCl275 μM), and the phenotype after stress was recorded by taking photos. The root length, plant height, fresh weight, dry weight and water content were counted. All experimental techniques were repeated for 3 times, and each time 10 plants of each line were used.

[0162] The results are shown in Figure 5 : Under control conditions, the growth of plants of each line was almost the same. After cadmium stress treatment, the plants were inhibited to a certain extent, but OE13 and OE15 were obviously more resistant than WT, and the plant height and root length were higher. In contrast, the mutant was more sensitive, and the plants were shorter and the roots were shorter. This indicates that SlJMJ gene is involved in the process of plant response to cadmium stress, thereby improving the tolerance of plants to cadmium stress.

[0163] II. Determination of physiological index of overexpression and mutant under cadmium treatment

[0164] 1. Effect of SlJMJ gene on photosynthetic system of tomato leaves under cadmium stress

[0165] To further verify the tolerance of SlJMJ overexpression and mutant tomato to cadmium stress, the contents of chlorophyll a, chlorophyll b, total chlorophyll and carotenoid of overexpression and mutant lines after 75 μM CdCl2 stress treatment were determined, and the results are shown in Figure 6 : Under no stress treatment, there was no significant difference in the contents of chlorophyll a, chlorophyll b, total chlorophyll and carotenoid between overexpression and mutant plants and WT plants. After stress treatment, the four index values of overexpression plants showed a significant upward trend compared with WT plants, and the mutant plants showed a significant downward trend compared with WT plants. Such results confirm that SlJMJ gene helps to alleviate the damage of Cd stress to the process of photosynthetic pigment biosynthesis, thereby enhancing the Cd tolerance of tomato.

[0166] 2. Effect of SlJMJ gene on antioxidant system of tomato leaves under cadmium stress

[0167] To further verify the tolerance of SlJMJ overexpression and mutant tomato to cadmium stress, the antioxidant enzyme content of overexpression and mutant lines was determined after 75 μM CdCl2stress treatment, and the results are shown in Table 2. Figure 7 As shown in Table 2, there was no significant difference in the contents of SOD, POD, CAT and APX between overexpression and mutant plants and WT plants without stress treatment. After cadmium stress, the four index values of overexpression lines increased significantly compared with WT plants, while the mutant lines decreased significantly. It is indicated that overexpression of SlJMJ gene can enhance the activity of antioxidant enzymes in plants. To further verify the effect of SlJMJ gene on the antioxidant system of tomato leaves under cadmium stress, NBT and DAB staining of tomato leaves was performed, and the contents of superoxide anion, hydrogen peroxide and MDA were measured, and the results are shown in Table 3. Figure 8 As shown in Table 3, there was no obvious difference in the leaves of WT, overexpression and mutant plants without stress treatment. After cadmium stress treatment, the mutant leaves had the deepest staining, and the overexpression leaves had the shallowest staining, indicating that overexpression of SlJMJ can alleviate the oxidative damage of tomato leaves. After stress, the contents of superoxide anion, hydrogen peroxide and MDA in the mutant were the highest, and the contents in the overexpression plants were the lowest, which also confirmed this result.

[0168] III. Determination of the expression amount of related Marker genes of overexpression and mutant under cadmium treatment

[0169] 1. Treatment of plant material

[0170] The SlJMJ transgenic overexpression tomato lines OE-13 and OE-15 and mutant lines jmj12-4 and jmj12-10 of T2 generation obtained by cutting were quickly frozen in liquid nitrogen and then stored at -80°C.

[0171] 2. Extraction of total RNA and obtaining of cDNA

[0172] The total RNA of the test tissue samples treated at different times obtained in step 1 above was extracted by using Plant Total RNA Isolation Kit kit (Foregene); and the cDNA was obtained by reverse transcription using the obtained total RNA as a template.

[0173] 3. Real-time PCR

[0174] The above cDNA was used as a template, and the alkali stress related Marker gene primers were used to detect the expression amount of SlJMJ gene by Real-time PCR. The primer sequences are as shown below:

[0175] Alkali stress related Marker genes: SlCAT1, SlPOD, SlSOD, SlGST, SlAPX1, SlAPX2.

[0176] SlCAT1-S: 5'-AAATGGGTTGAGTCTTTATCCGA-3' (SEQ ID NO. 14);

[0177] SlCAT1-AS: 5'-TCATTGATTTTTCACATTGTAGGCT-3' (SEQ ID NO. 15).

[0178] SlPOD-S: 5'-CTTGCCCTAATGCTCTCACC-3' (SEQ ID NO. 16);

[0179] SlPOD-AS: 5'-GCATCACAACCCTGAACAAA-3' (SEQ ID NO. 17).

[0180] SlSOD-S: 5'-GTCCACAGTCCATCATTGGAAGA-3' (SEQ ID NO. 18);

[0181] SlSOD-AS: 5'-ATAGAAAACAAGGCTCAGCAGCT-3' (SEQ ID NO. 19).

[0182] SlGST-S: 5'-CTCTGGTTTGGAGCAATTCA-3' (SEQ ID NO. 20);

[0183] SlGST-AS: 5'-AATTTCAGCTGGATGCCTTT-3' (SEQ ID NO. 21).

[0184] SlAPX1-S: 5'-CTGATGTTCCCTTTCACCCTG-3' (SEQ ID NO. 22);

[0185] SlAPX1-AS: 5'-ATTTCAATAGAAGTTCCCAGTAGCA-3' (SEQ ID NO. 23).

[0186] SlAPX2-S: 5'-TCAGTGATCCTGCTTTCCGC-3' (SEQ ID NO. 24);

[0187] SlAPX2-AS: 5'-TGTCACCACCCTCCCAACTCT-3' (SEQ ID NO. 25).

[0188] Real-time PCR reaction conditions: 94℃ 10min → [94℃ 30s → 59℃ 30s → 72℃ 45s] × 40 → 72℃ 10min → 94℃ 30s.

[0189] Real-time PCR used the comparative CT method (ΔΔCT) to calculate gene expression levels, with the tomato SlActin11 gene as the internal reference and untreated samples as the control. Differences in target gene expression were expressed as the fold increase in the expression of treated samples relative to untreated samples at each time point. Each sample included three biological replicates and three technical replicates; the data were the average of the three biological replicates, and if one value showed a large deviation, the average of the two data points was used. Raw data were standardized. The standardized data were then analyzed for significance using a T-test. Relative expression level 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 primer sequences for the internal reference gene are shown below:

[0190] Actin11-S: 5'-AAGATCCCATTCGTCCCCAT-3' (SEQ ID NO. 26);

[0191] Actin11-AS: 5'-CAAGAGCCTCAAGGAGAGTTGG-3' (SEQ ID NO. 27).

[0192] The results are as follows Figure 9 As shown, after treatment with 75 μM CdCl2 for 6 h, the relative expression levels of six alkali stress-related marker genes in WT and SlJMJ transgenic tomatoes were analyzed. The relative expression levels of all six genes in overexpressing tomatoes OE-13 and OE-15, as well as mutant tomatoes jmj-4 and jmj-10, were significantly different from those in WT after stress treatment. Specifically, the expression levels of SlCAT1, SlPOD, SlSOD, SlAPX1, and SlAPX2 in OE-13 and OE-15 were significantly upregulated compared to WT, while those in jmj-4 and jmj-10 were significantly downregulated. Compared to WT, the expression level of SlGST in OE-13 and OE-15 was significantly downregulated after stress treatment, while the expression level in jmj-4 and jmj-10 was significantly upregulated. The above results indicate that under cadmium stress, the expression levels of stress marker genes such as SlCAT1, SlPOD, and SlSOD in SlJMJ12 overexpressing tomatoes and mutant tomatoes are significantly different from those in WT tomatoes. Therefore, the SlJMJ gene may enhance the tolerance of tomatoes to cadmium stress by regulating the expression levels of stress-induced marker genes.

Claims

1. Application of a protein with the amino acid sequence shown in SEQ ID NO.28 in enhancing the cadmium resistance of tomatoes.

2. Application of the gene with the nucleotide sequence shown in SEQ ID NO.3 in improving the cadmium resistance of tomatoes.

3. Application of recombinant vectors containing genes with nucleotide sequences as shown in SEQ ID NO.3 in improving the cadmium resistance of tomatoes.

4. The application according to claim 3, characterized in that, The starting vector for the recombinant vector was pCAMBIA2300.

5. Application of recombinant microbial cells containing genes with nucleotide sequences as shown in SEQ ID NO.3 in enhancing the cadmium resistance of tomatoes.

6. The application according to claim 5, characterized in that, The recombinant microbial cells are eukaryotic or prokaryotic microbial cells.

7. Application of tomato plants overexpressing the gene with the nucleotide sequence shown in SEQ ID NO.3 in improving cadmium resistance.

8. A method for improving the cadmium resistance of tomatoes, characterized in that, The specific steps of the method are as follows: Step 1: Ligate the gene with the nucleotide sequence shown in SEQ ID NO.3 to the vector to obtain the recombinant vector; Step 2: The recombinant vector obtained in Step 1 is transferred 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, The carrier mentioned in step 1 is pCAMBIA2300.

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