Application of cadmium-resistant gene in improvement of cadmium resistance of tomato

Abstract

The invention discloses application of a cadmium-resistant gene to 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 discloses application of a protein with an amino acid sequence as shown in SEQ ID NO.28 in improvement of cadmium resistance of tomatoes. The SlJMJ gene enhances the tolerance to cadmium stress by adjusting the expression level of an adversity stress induction marker gene.

Description

Technical Field

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

[0002] Cadmium (Cd) is one of the most harmful and widely distributed toxic heavy metal pollutants in agricultural soil. It has extremely strong biological persistence and will exist for a long time after being consumed by organisms. The investigation and assessment of the Ministry of Ecology and Environment found that heavy metal pollution such as cadmium in cultivated soil mainly comes from non-ferrous metal smelting, mining and other industries, and enters the soil through hydraulic transport or atmospheric deposition; pollutants related to industrial and mining enterprises mainly come from chemical, petrochemical, coking and other industries, and toxic and harmful substances enter the soil through leakage, loss and dispersion. Due to rapid industrialization and urbanization, there are huge challenges in protecting cultivated soil from heavy metal pollution. The use of grains, vegetables and rhizomes grown in contaminated soil has caused concerns about food security and human health. From the perspective of pollutant exceeding the standard, the point exceeding rate of cadmium is 7.0%, ranking first among common inorganic pollutants.

[0003] Tomato is one of the most consumed vegetables in daily life, and its quality directly affects people's daily food safety. 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 the excessive accumulation of cadmium in tomatoes may pose a great threat to human health. Therefore, finding a method for tomato to resist cadmium stress is a technical problem that needs to be solved urgently. Summary of the invention

[0004] The purpose of the invention is to provide a method for tomato to resist cadmium stress.

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

[0006] The present invention provides an application of a nucleic acid molecule such as a gene shown in SEQ ID NO.3 in improving the cadmium resistance of tomatoes.

[0007] The invention provides an application of a recombinant vector containing a nucleic acid molecule such as a gene shown in SEQ ID NO.3 in improving the cadmium resistance of tomatoes.

[0008] It is further defined that the starting vector of the recombinant vector is pCAMBIA2300.

[0009] The present invention provides an application of a recombinant microbial cell containing a gene shown by a nucleic acid molecule such as SEQ ID NO.3 in improving the cadmium resistance of tomatoes.

[0010] It is further defined that the recombinant microbial cell is a eukaryotic microbial cell or a prokaryotic microbial cell.

[0011] The present invention provides a use of a tomato plant over-expressing a gene represented by a nucleic acid molecule such as SEQ ID NO.3 in improving cadmium resistance.

[0012] The present invention provides a method for improving the cadmium resistance of tomatoes, and the specific steps of the method are as follows:

[0013] Step 1: Connect the nucleic acid molecule such as the gene shown in SEQ ID NO.3 to a vector to obtain a recombinant vector;

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

[0015] Step 3: Infect tomatoes with the recombinant Agrobacterium obtained in step 2 to obtain transgenic tomatoes.

[0016] It is further defined that the primers for amplifying the gene represented by the nucleic acid molecule as SEQ ID NO.3 in step 1 are SEQ ID NO.1 and SEQ ID NO.2.

[0017] It is further defined that the vector in step 1 is pCAMBIA2300.

[0018] Beneficial effect: A tomato cadmium-resistant gene sequence is shown in SEQ ID NO.3, and an overexpression vector and a mutant thereof are constructed. Through the tomato homologous transformation method mediated by Agrobacterium rhizogenes, a tomato overexpressing the gene shown in SEQ ID NO.3 and a sljmj mutant tomato are successfully obtained. The experiment of the present invention proves that overexpressing the gene in tomatoes can enhance the tolerance of tomatoes to cadmium stress at the seedling stage, and the sljmj mutant shows lower tolerance to cadmium stress. It is shown that the gene can lay a foundation for the research on cultivating transgenic plants with cadmium resistance. After cadmium stress treatment, the plant phenotype, dry weight, fresh weight and related physiological indicators were analyzed. The results showed that compared with the control, the plant phenotype of SlJMJ-OE tomatoes was better, with larger values ​​such as root length, plant height and fresh weight, while the plant phenotype of sljmj (jmj) mutant tomatoes was the worst, with smaller values ​​such as root length, plant height and fresh weight; SlJMJ-OE (JMJ-OE) had lower H2O2 content and higher antioxidant enzyme activities such as SOD, POD, CAT, APX, while the sljmj mutant had the opposite. Such research results confirm that the SlJMJ gene can enhance the resistance of tomatoes to cadmium stress. The expression of stress-related marker genes in transgenic tomatoes was analyzed, and it was found that under cadmium stress, stress marker genes such as SlCAT1, SlPOD, and SlSOD were up-regulated in SlJMJ-OE transgenic tomatoes, and the expression levels were significantly higher than those in WT, while they were down-regulated in sljmj mutant tomatoes, and the expression levels were significantly lower than those in WT. Therefore, the SlJMJ gene enhances tolerance to cadmium stress by regulating the expression levels of stress-induced marker genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 PCR amplification of SlJMJ gene;

[0020] Figure 2 This is the PCR detection image of SlJMJ overexpression tomato positive seedlings;

[0021] Figure 3 This is a diagram for the identification of RNA levels in SlJMJ overexpressing tomato;

[0022] Figure 4 This is the identification picture of SlJMJ gene mutation tomato positive seedlings;

[0023] Figure 5 The phenotypes, plant height and root length of SlJMJ overexpression and mutant tomatoes under cadmium stress;

[0024] Figure 6 is the photosynthetic pigment content of SlJMJ transgenic tomatoes under cadmium stress;

[0025] Figure 7 is the antioxidant enzyme activity of SlJMJ transgenic tomatoes under cadmium stress;

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

[0027] Fig. 9 This is an analysis of marker gene expression related to cadmium stress. DETAILED DESCRIPTION

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0029] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0030] The DH5α competent cells, Agrobacterium GV3101 competent cells, and Agrobacterium K599 competent cells in the following examples are products of Quanshijin Company.

[0031] The Micro-Tom tomatoes used in the following examples are commercially available.

[0032] Example 1. Cloning of the tomato S1JMJ gene

[0033] 1. Processing of plant materials

[0034] Select plump Micro-Tom tomato seeds, rinse them with sterile water 3-4 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 pots filled with nutrient soil (peat soil: vermiculite = 2:1) 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 4 leaves and 1 heart, take the leaves and put them in EP tubes and store them at -80℃.

[0035] 2. RNA Extraction

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

[0037] 3. Obtaining cDNA

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

[0039] 4. PCR amplification

[0040] 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:

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

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

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

[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; terminate the reaction at 4°C.

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

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

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

[0048] ATGGATGATATACCTGAATGGCTTAAAGGGTTGCCCTCGCACCTGAATTTCGACCTACT

[0049] GATACTGAATTTGCTGACCCATTGCCTATATATCAAAAATCGAGAAGGAAGCTAGTGCT

[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] GCTGGTTTGCTTGGCATGTTGAGGATCATGAGCTTCACAGTCTAAATTTTTCTTCATACAG

[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 SlJMJ-OE plants

[0072] 1. Obtaining SlJMJ overexpression recombinant plasmid

[0073] 1. Acquisition of SlJMJ gene

[0074] Using pCAMBIA2300-SlJMJ 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 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] PCR amplification system (25 μl): cDNA 1 μl, Primer-F 1 μl, Primer-R 1 μl, Prime Star Mix 12.5 μl, ddH2O 9.5 μl.

[0078] PCR amplification conditions: 98°C for 10 s, 58°C for 10 s, 72°C for 1 min 30 s, 35 cycles; 72°C for 5 min; terminate the reaction at 4°C.

[0079] 2. Obtaining plant expression vector

[0080] The pCAMBIA 2300 vector and the PCR amplification product were digested with restriction endonucleases and ligated to obtain the recombinant vector pCAMBIA 2300-SlJMJ, which was then sequenced and verified.

[0081] The sequencing results showed that the recombinant vector pCAMBIA 2300-SlJMJ was obtained by replacing the DNA fragment between the restriction sites of the pCAMBIA 2300 vector with the SlJMJ gene shown in sequence 1 in the sequence table, while keeping the other sequences of the pCAMBIA2300 vector unchanged.

[0082] 3. Conversion

[0083] The recombinant vector pCAMBIA 2300-SlJMJ was transformed into Agrobacterium tumefaciens GV3101 by heat shock method, and positive transformants were obtained by PCR identification and used to infect tomato plants.

[0084] II. Obtaining SlJMJ Overexpression and Mutant Tomato Plants

[0085] 1. Wash the seeds

[0086] Select plump tomato seeds, soak them at 55℃ for 15 minutes, disinfect them with 75% ethanol for 30 seconds, rinse them with sterile water 2-3 times, 2 minutes each time, disinfect them with 2% sodium hypochlorite solution for 15 minutes, and rinse them with sterile water 6-7 times, 2 minutes each time.

[0087] 2. Vaccination

[0088] In the clean bench, place the sterilized seeds on sterilized filter paper, absorb the moisture, inoculate the seeds into the sterilized 1 / 2MS solid culture medium, place the tissue culture bottle in the tissue culture room for dark culture, and when the seeds show white spots, place them under normal light culture conditions (16h light / 8h dark) for culture.

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

[0090] 3. Pre-cultivation

[0091] The seedlings grown from the flattened cotyledons of the sterile seedlings were cut. The sterile seedlings were clipped out with tweezers and placed in a glass culture dish lined with filter paper. The cotyledons were cut into pieces of about 0.5 cm × 0.5 cm in size, with the back of the cotyledons facing downwards, and placed in the preculture medium, and cultured under normal light culture conditions 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-cultivation

[0094] Prepare sterilized beakers, filter screens, filter papers, glass culture dishes, and tweezers in advance.

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

[0096] Use tweezers to gently pick up the pre-cultured explants, put them into a beaker, pour in the bacterial solution, and after contact with the bacterial solution for 10 minutes, put the explants back into the pre-culture medium and culture them in the dark for 2 days with the back of the cotyledons facing upwards.

[0097] 5. Screening

[0098] After co-cultivation, the cotyledons are transferred to the selection medium with the back of the cotyledons facing downward. When the cotyledons swell and grow green buds, they are transferred to a new medium for further screening until callus sprouts.

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

[0100] 6. Rooting

[0101] When adventitious buds grow out, the callus tissue is cut off, and 2-3 cm buds are cut off and inoculated into the rooting medium.

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

[0103] 7. Domestication

[0104] Select tissue culture seedlings with strong roots, 4-6cm seedling height and vigorous growth, half open the bottle cap to harden the seedlings for 2 days, remove the tissue culture seedlings from the culture medium, and wash the culture medium remaining on the roots. Mix peat soil and vermiculite in a ratio of 2:1, place in a flowerpot, water it to saturate it, and transplant the clean rooted seedlings into it. After transplanting, cover with film to keep it moist, place it in a greenhouse at 25℃, and open the film after 5-7 days to start normal management.

[0105] 3. Identification of overexpression positive strains

[0106] 1. Identification of positive seedlings of SlJMJ overexpressing tomatoes

[0107] Take 0.1 g of leaves from the T0 generation SlJMJ overexpressing seedlings, extract DNA, and perform PCR identification. The specific steps are as follows:

[0108] DNA of the T0 generation SlJMJ overexpressing plants obtained after domestication was extracted, and the DNA was used as a template to design primers using the sequence connected to the 35S promoter and the target gene SlJMJ, and PCR detection was performed. The primer sequences are as follows:

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

[0110] 35S / J12-R: 5'-AACAAAGAAGAGGATGGTGTCTTCTAC-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°C 2 min → [94°C 15 s → 58°C 30 s → 72°C 1 min 30 s] × 35 cycles → 72°C 10 min → 4°C to terminate the reaction;

[0113] The PCR amplification products were subjected to 1% agarose gel electrophoresis, and the detection results were as follows Figure 2 As shown: the transgenic plants can amplify a specific band of 1042 bp, while the wild-type plants cannot amplify a specific band, and there are 18 resistant seedlings that are positive in PCR detection.

[0114] 2. Identification of RNA levels in SlJMJ-overexpressing tomatoes

[0115] Take the tomato seedlings that are positive in PCR test and perform fluorescence quantitative PCR. The specific steps are as follows:

[0116] Take 0.05g of leaves from tomato seedlings that are positive in PCR test, extract RNA and reverse transcribe it into cDNA, and perform fluorescence quantitative PCR. The primer sequences 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]×40→72℃10min→95℃30s

[0120] The results are as follows Figure 3 As shown: The results showed that the expression level of SlJMJ gene in OE13 strain was the highest, about 21 times that of WT plants; the expression levels of SlJMJ gene in OE15 and OE18 strains were also high, about 19 and 18.2 times that of WT plants, respectively. This shows that the SlJMJ gene overexpression tomato SlJMJ-OE was successfully obtained. In addition, OE13 and OE15 strains with higher gene expression levels were selected for subsequent experiments.

[0121] Example 3. Obtaining SlJMJ mutant plants

[0122] 1. Obtaining the SlJMJ mutant recombinant plasmid

[0123] 1. Using the CRISPR-P website of Huazhong Agricultural University (http: / / crispr.hzau.edu.cn / CRISPR2 / ), two targets were selected in the CDS region of the SlJMJ gene and in the front end of the protein or the important functional domain region as much as possible. The target sequences are as follows: Target1: 5'-ACCCAATTCCTGATGTCGGGTGG-3' (SEQ ID NO.10); Target2: 5'-CATACTCGACGTAGATAGGGTGG-3' (SEQ ID NO.11), and both targets were connected to the CRISPR / Cas9 knockout vector. After the design, the SlJMJ gene CRISPR / Cas9 knockout vector was constructed by Jiangsu Weimi Biotechnology Co., Ltd.

[0124] 2. Conversion

[0125] The mutant plasmid was transformed into Agrobacterium tumefaciens GV3101 by heat shock method, and positive transformants were obtained by PCR identification and 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°C for 2 min, [94°C for 30 s, 58°C for 30 s, 72°C for 30 s] × 35 cycles; 72°C for 10 min; terminate the reaction at 4°C.

[0130] II. Obtaining SlJMJ Overexpression and Mutant Tomato Plants

[0131] 1. Wash the seeds

[0132] Select plump tomato seeds, soak them at 55℃ for 15 minutes, disinfect them with 75% ethanol for 30 seconds, rinse them with sterile water 2-3 times, 2 minutes each time, disinfect them with 2% sodium hypochlorite solution for 15 minutes, and rinse them with sterile water 6-7 times, 2 minutes each time.

[0133] 2. Vaccination

[0134] In the clean bench, place the sterilized seeds on sterilized filter paper, absorb the moisture, inoculate the seeds into the sterilized 1 / 2MS solid culture medium, place the tissue culture bottle in the tissue culture room for dark culture, and when the seeds show white spots, place them under 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-cultivation

[0137] The seedlings grown from the flattened cotyledons of the sterile seedlings were cut. The sterile seedlings were clipped out with tweezers and placed in a glass culture dish lined with filter paper. The cotyledons were cut into pieces of about 0.5 cm × 0.5 cm in size, with the back of the cotyledons facing downwards, and placed in the preculture 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-cultivation

[0140] Prepare sterilized beakers, filter screens, filter papers, glass culture dishes, and tweezers in advance.

[0141] Start preparing Agrobacterium: take 1 mL of the preserved glycerol bacteria and transfer it to 100 mL LB liquid culture medium with Rif and Kan, culture it overnight at 28°C, 200 rpm, centrifuge it at 5000 rpm for 5 min, pour out the supernatant after centrifugation, add liquid MS to resuspend it, the OD is about 0.6 (diluted bacterial solution), 50 mL bacterial solution + 50 mL MS.

[0142] Use tweezers to gently pick up the pre-cultured explants, put them into a beaker, pour in the bacterial solution, and after contact with the bacterial solution for 10 minutes, put the explants back into the pre-culture medium and culture them in the dark for 2 days with the back of the cotyledons facing upwards.

[0143] 5. Screening

[0144] After co-cultivation, the cotyledons are transferred to the selection medium with the back of the cotyledons facing downward. When the cotyledons swell and grow green buds, they are transferred to a new medium for further screening until callus sprouts.

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

[0146] 6. Rooting

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

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

[0149] 7. Domestication

[0150] Select tissue culture seedlings with strong roots, 4-6cm seedling height and vigorous growth, half open the bottle cap to harden the seedlings for 2 days, remove the tissue culture seedlings from the culture medium, and wash the culture medium remaining on the roots. Mix peat soil and vermiculite in a ratio of 2:1, place in a flowerpot, water it to saturate it, and transplant the clean rooted seedlings into it. After transplanting, cover with film to keep it moist, place it in a greenhouse at 25℃, and open the film after 5-7 days to start normal management.

[0151] 3. Identification of positive mutant tomato seedlings

[0152] Take 0.1 g of leaves from the T0 generation SlJMJ mutant seedlings, extract DNA, and perform PCR identification. The specific steps are as follows:

[0153] The DNA of the T0 generation SlJMJ mutant plants obtained after domestication was extracted, and PCR detection was performed using the DNA as a template and Cas9-F / R primers. 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] × 35 cycles; 72°C for 10 min; terminate the reaction at 4°C.

[0158] The PCR amplification products were subjected to 1% agarose gel electrophoresis, and the detection results were as follows Figure 4 As shown: the mutant plants can amplify a specific band of 478bp, while the wild-type plants do not amplify a specific band. Among them, 28 jmj mutants were resistant seedlings that tested positive by PCR.

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

[0160] 1. Determination of growth indexes of overexpression and mutants at seedling stage under cadmium treatment

[0161] The seeds of wild-type tomato WT, SlJMJ overexpression T2 homozygous lines OE13 and OE15, and mutant T2 homozygous lines jmj12-4 and jmj12-10 were washed with sterile water 3 to 4 times and placed on moist filter paper. They were cultured in the dark at 25°C for 4 days to germinate. When the buds grew to about 1 to 2 cm, they were transferred to pots filled with nutrient soil (peat soil: vermiculite = 2:1) and placed in a greenhouse for culture. The growth conditions in the greenhouse were 25°C / 20°C (day / night) and the photoperiod was 16 hours of light / 8 hours of darkness. When the seedlings grew to four leaves and one heart, they were transferred to aqueous solution for stress treatment (CdCl2 75μM), and the phenotypes after stress were recorded by taking photos, and the root length, plant height, fresh weight, dry weight and water content were counted. All experiments were repeated 3 times for technical and biological replications, and 10 plants were tested for each line in each experiment.

[0162] The results are as follows Figure 5 As shown in the figure: Under control conditions, the growth of plants in 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, with higher plant height and root length, while the mutants were more sensitive, with shorter plants and shorter roots. This shows that the SlJMJ gene is involved in the process of plant response to cadmium stress, thereby improving the tolerance of plants to cadmium stress.

[0163] 2. Determination of physiological indicators of overexpression and mutants at seedling stage under cadmium treatment

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

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

[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 tomatoes to cadmium stress, the antioxidant enzyme content of overexpression and mutant strains was measured after 75 μM CdCl2 stress treatment. Figure 7 As shown in the figure: When no stress treatment was applied, there was no significant difference in the content of SOD, POD, CAT and APX between the overexpression and mutant plants and the WT plants. After cadmium stress was applied, the values ​​of the four indicators of the overexpression strains increased significantly compared with the WT plants, while those of the mutant strains decreased significantly. This indicates that overexpression of the SlJMJ gene will enhance the activity of the plant's antioxidant enzymes. In order to further verify the effect of the SlJMJ gene on the antioxidant system of tomato leaves under cadmium stress, the tomato leaves were stained with NBT and DAB, and the contents of superoxide anions, hydrogen peroxide and MDA were measured. The results are shown in the figure. Figure 8 As shown in the figure: When no stress was applied, there was no obvious difference between the leaves of WT, overexpression and mutant. After cadmium stress, the leaves of mutant were the darkest and the leaves of overexpression were the lightest, indicating that SlJMJ overexpression can alleviate the oxidative damage of tomato leaves. After stress, the mutant had the highest content of superoxide anion, hydrogen peroxide and MDA, while the overexpression plant had the lowest content, which also confirmed this result.

[0168] 3. Determination of related marker gene expression levels in overexpressed and mutant strains under cadmium treatment

[0169] 1. Processing of plant materials

[0170] The plant materials obtained were cut and the T2 generation SlJMJ transgenic overexpressing tomato lines OE-13 and OE-15, and the mutant lines jmj12-4 and jmj12-10 were used as tissue samples to be tested, quickly frozen in liquid nitrogen, and then stored at -80°C.

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

[0172] The total RNA of the tissue samples to be tested obtained in step 1 after being treated for different time periods was extracted using the Plant Total RNA Isolation Kit (Foregene). The total RNA was used as a template for reverse transcription to obtain cDNA.

[0173] 3. Real-time PCR

[0174] Using the above cDNA as a template and alkaline stress-related marker gene primers, the expression level of the SlJMJ gene was detected by Real-time PCR. The primer sequences are as follows:

[0175] Marker genes related to alkali stress: 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°C 10 min→[94°C 30 s→59°C 30 s→72°C 45 s]×40→72°C 10 min→94°C 30 s.

[0189] Real-time PCR uses the comparative CT method (△△CT) to calculate gene expression, with the 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:

[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 Fig. 9 As shown: After 6h of treatment with 75μM CdCl2, the relative expression levels of six alkaline stress-related marker genes in WT and SlJMJ transgenic tomatoes were analyzed. The relative expression levels of the six genes in overexpressed tomatoes OE-13 and OE-15 and mutant tomatoes jmj-4 and jmj-10 after stress treatment were significantly different from those in WT. Among them, the expression levels of SlCAT1, SlPOD, SlSOD, SlAPX1, and SlAPX2 in OE-13 and OE-15 were significantly upregulated compared with WT, while those in jmj-4 and jmj-10 were significantly downregulated; compared with WT, the expression levels of SlGST in OE-13 and OE-15 were significantly downregulated after stress treatment, while those in jmj-4 and jmj-10 were significantly upregulated. The above results indicate that under cadmium stress conditions, the expression levels of stress marker genes such as SlCAT1, SlPOD, and SlSOD in SlJMJ12 overexpressing tomatoes and mutant tomatoes were significantly different from those in WT. 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 having an amino acid sequence as shown in SEQ ID NO.28 in improving the cadmium resistance of tomatoes.

2. Application of the gene represented by nucleic acid molecule SEQ ID NO.3 in improving the cadmium resistance of tomatoes.

3. Use of a recombinant vector containing a nucleic acid molecule such as the gene shown in SEQ ID NO.3 in improving the cadmium resistance of tomatoes.

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

5. Use of recombinant microbial cells containing a nucleic acid molecule such as a gene shown in SEQ ID NO.3 in improving the cadmium resistance of tomatoes.

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 tomato plants overexpressing a nucleic acid molecule such as a gene shown in SEQ ID NO. 3 in improving cadmium resistance.

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 nucleic acid molecule such as the gene shown in SEQ ID NO.3 to a 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 gene represented by the nucleic acid molecule as SEQ ID NO.3 are SEQ ID NO.1 and SEQ ID NO.

2.

10. The method according to claim 8, characterized in that The vector described in step 1 is pCAMBIA2300.

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