SlHZ41 Gene and Its Application in Improving Heat Tolerance and Fruit Size of Tomato

Abstract

The present invention belongs to the technical field of genetic engineering, and particularly relates to the SlHZ41 gene and its application in improving the heat tolerance and fruit size of tomatoes. The nucleotide sequence of the SlHZ41 gene provided by the present invention is shown in SEQ ID NO.1, and the amino acid sequence of the protein expressed by it is shown in SEQ ID NO.2. The knockout vector of the SlHZ41 gene provided by the present invention can be used to improve the activities of POD and SOD in plants, inhibit the accumulation of MDA content, and reduce the damage of high-temperature stress to plants; at the same time, the knockout plants increase the yield of tomatoes; it provides a theoretical basis for exploring the resistance mechanism, field yield and gene utilization of the SlHZ41 gene.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to the SlHZ41 gene and its application in improving the heat tolerance and fruit size of tomatoes. Background Art

[0002] Tomato (Solanum lycopersicum L.) is an important vegetable crop widely cultivated around the world, rich in vitamin C, lycopene, dietary fiber, potassium, β-carotene, etc., and has functions such as antioxidant, protecting heart health, preventing cancer, protecting eyesight, and anti-inflammatory. Statistical data from the Food and Agriculture Organization of the United Nations database (FAOSTAT) shows that: in 2022, the planting area of tomatoes in China reached 1.137 million hectares, and the output reached 68.24 million tons, accounting for nearly 1 / 3 of the world's output.

[0003] Tomatoes are sensitive to environmental temperature. In China, the planting is mainly carried out in simple greenhouse greenhouses and open fields, with relatively rough management, and is easily damaged by various stress hazards such as high temperature. Among them, high temperature has become one of the important limiting factors in tomato production, often causing huge economic losses. To ensure the stable and sustainable development of the tomato industry, it is necessary to give priority to the development of breeding, utilize modern biotechnology to explore various genetic resources, and vigorously develop gene editing technology to improve breeding efficiency. In terms of breeding goals, in addition to continuing to focus on commercial quality, it is also necessary to accelerate the breeding of stress-resistant varieties that are resistant to high temperature and other adverse conditions.

[0004] The homeodomain-leucine zipper (HD-Zip) transcription factor family is a class of transcription factors unique to plants, which are widely involved in the processes of plant growth and development and stress response. Family members generally have the characteristic of being induced to express under stress, and participate in regulating hormone signals and various stress responses. The expressions of Arabidopsis thaliana AtHB5, AtHB6, AtHB7, and AtHB12 are induced by drought and abscisic acid treatment. Overexpression of Medicago sativa MsHB7 reduces the salt stress tolerance of transgenic plants. Tomato SlHB15A can bind to the SlIAA32 promoter to regulate auxin signaling, thereby regulating tomato pedicel abscission. Tomato HD-Zip subfamily member 8 is involved in tomato trichome development. However, the role of tomato HD-Zip in the regulation of high temperature tolerance and fruit size has rarely been reported. Summary of the Invention

[0005] To solve the problem that there are few reports on the role of tomato HD-Zip in the regulation of high temperature tolerance and fruit size in the prior art, the present invention adopts the following technical solutions:

[0006] The inventors found that SlHZ41 was induced by heat when screening HD-Zip I subfamily members in preliminary experiments, and found that the change in the expression level of the SlHZ41 gene can significantly affect the heat resistance and reproductive growth of the plant, showing the characteristics of a "negative regulatory factor" for high temperature stress response and fruit size, and has great potential application value. Based on this, the present invention provides a SlHZ41 gene, the nucleotide sequence of the SlHZ41 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein expressed by it is shown in SEQ ID NO.2.

[0007] The present invention also provides application of the S1HZ41 gene in improving the heat resistance and fruit size of tomatoes.

[0008] Specifically, the SlHZ41 gene was knocked out by using the genetic engineering method CRISPR / Cas9 gene editing technology, thereby obtaining a tomato mutant with improved heat resistance and fruit size.

[0009] The nucleotide sequence of the SlHZ41 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein expressed by it is shown in SEQ ID NO.2;

[0010] The sequence of the target site of the S1HZ41 gene is shown in SEQ ID NO.3.

[0011] Preferably, the target site of the S1HZ41 gene is connected to a gene expression vector; the expression vector is transfected into the plant material to be transformed to obtain a tomato knockout plant with site-directed editing of S1HZ41. The S1HZ41 knockout plant has enhanced heat tolerance, and increased fruit size and fruit yield.

[0012] Preferably, the transgenic vector comprises SlHZ41-pHellsgate2 and / or SlHZ41-Cas9-pCAMBIA2301 and / or SlHZ41-1305 and / or SlHZ41-pHSE401.

[0013] The present invention also provides a method for cultivating heat-resistant tomatoes with improved fruit size, comprising the following steps:

[0014] Obtaining the target site sequence at the nucleotide sequence of the S1HZ41 gene, and synthesizing an sgRNA primer for the target site sequence;

[0015] The sgRNA primers were used to construct the SlHZ41-Cas9-pCAMBIA2301 vector;

[0016] Transfer the vector containing SlHZ41-Cas9-pCAMBIA2301 into Agrobacterium to obtain recombinant Agrobacterium; Transfect the recombinant Agrobacterium into the tomato to be transformed, and through selection, differentiation, rooting, acclimatization, and transplantation, obtain heat-tolerant and fruit size-improved tomato plants.

[0017] Preferably, the sgRNA primer sequences include SlHZ41-SgRNA-F and SlHZ41-SgRNA-R;

[0018] The nucleotide sequence of SlHZ41-SgRNA-F is as shown in SEQ ID NO.4; SlHZ41-SgRNA-R is as shown in SEQ ID NO.5.

[0019] Preferably, after transfecting the recombinant Agrobacterium into the tomato to be transformed, obtain transgenic positive seedlings of tomato SlHZ41 through tissue culture, obtain the SlHZ41 gene fragment containing the target site through PCR amplification, and sequence to confirm to obtain tomato plants with SlHZ41 gene mutations.

[0020] Among them, the primers used for the PCR amplification include SlHZ41-check-F and SlHZ41-check-R.

[0021] The nucleotide sequence of SlHZ41-check-F is as shown in SEQ ID NO.6, and the nucleotide sequence of SlHZ41-check-R is as shown in SEQ ID NO.7.

[0022] Since the resistance mechanism of plants to stress is very complex, improving the stress resistance of crops by traditional breeding methods often takes a long time and consumes a large amount of human and material resources; Combining traditional breeding methods with modern molecular biotechnology to explore and utilize resistance genes and eliminate harmful genes will greatly improve breeding efficiency and obtain better social and economic benefits. The gene site-directed editing (CRISPR / Cas) technology can accurately knock out harmful genes, and exogenous components such as vectors can be eliminated by genetic means, with the characteristics of both safety and efficiency, becoming a research hotspot in the current fields of gene function and crop improvement breeding.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides a SlHZ41 gene and its application in improving the heat tolerance and fruit size of tomatoes. The knockout vector of the SlHZ41 gene provided by the present invention can improve the activities of POD and SOD in plants, inhibit the accumulation of MDA content, and reduce the damage of high-temperature stress to plants; At the same time, the knockout plants increase the yield of tomatoes; It provides a theoretical basis for exploring the resistance mechanism, field yield and gene utilization of the SlHZ41 gene. Brief Description of the Drawings

[0025] Figure 1 This shows the expression of the SlHZ41 gene in different tissues of the present invention; where Rt: root; St: stem; YL: young leaf; OL: old leaf; LB: lateral branch; FL: flower; MG: green mature; B: breaker; B+3: 3 days after breaker; B+7: 7 days after breaker; B+10: 10 days after breaker; B+15: 15 days after breaker.

[0026] Figure 2 This shows the expression pattern of the SlHZ41 gene under high temperature stress in the present invention.

[0027] Figure 3 This shows the construction process of the SlHZ41-Cas9-pCAMBIA2301 vector in the present invention; where Figure 3 Figure A in it is the linearized PSgR-Cas9-At vector; Figure 3 Figure B in it is the PCR detection result of SlHZ41-SgRNA-Cas9; Figure 3 Figure C in it is the double digestion result of SlHZ41-SgRNA-Cas9; Figure 3 Figure D in it is the digestion result of pCAMBIA2301; Figure 3 Figure E in it is the detection result of SlHZ41-Cas9-pCAMBIA2301 in Escherichia coli.

[0028] Figure 4 This shows the construction process of the pHellsgate2-SlHZ41 overexpression vector in the present invention; where Figure 4 Figure A in it is the PCR amplification product of the SlHZ41 gene; Figure 4 Figure B in it is the double digestion result of pHellsgate2; Figure 4 Figure C in it is the detection result of pHellsgate2-SlHZ41 in Escherichia coli; Figure 4 Figure D in it is the detection result of the pHellsgate2-SlHZ41 recombinant plasmid in Agrobacterium tumefaciens.

[0029] Figure 5 This shows the detection result of the SlHZ41 gene knockout plants in the present invention; where Figure 5 Figure A in it is the structure of the SlHZ41 gene and the target site location; Figure 5 Figure B in it is the editing method of the SlHZ41 knockout site.

[0030] Figure 6 This shows the detection result of the SlHZ41 gene overexpression plants in the present invention.

[0031] Figure 7In the present invention, the knockout of the SlHZ41 gene significantly improves the heat tolerance of plants; among them, Figure 7 Figure (A) in Figure 7 is the phenotype after high-temperature treatment. Among them, the five columns of plants from left to right correspond to different plant lines in sequence. The plant lines corresponding from left to right are WT, OE1, OE2, KO1, and KO2; the plants corresponding in parallel in the upper and lower rows are plants of the same line; the upper row is the control group, which is the phenotype of each plant line without high-temperature treatment; the lower row is the phenotype of each plant line after high-temperature treatment; Figure 7 Figure (B) in Figure 7 is DAB staining; Figure 7 Figure (C) in Figure 7 is SOD activity; Figure 7 Figure (D) in Figure 7 is the relative conductivity; Figure 7 Figure (E) in Figure 7 is the malondialdehyde content.

[0032] Figure 8 In the present invention, the knockout of the SlHZ41 gene promotes fruit ripening and fruit size; among them, Figure 8 Figure A in Figure 8 is the fruit phenotypes of the second and third spikes of SlHZ41 transgenic plants and wild plants 3 months after transplantation. The plant lines corresponding from left to right are WT, OE1, OE2, KO1, and KO2; Figure 8 Figure B in Figure 8 is the phenotypes of the whole plants of SlHZ41 transgenic plants and wild plants 3 months after transplantation. The plant lines corresponding from left to right are WT, OE1, OE2, KO1, and KO2; Figure 8 Figure C in Figure 8 is the fruit quality of the second and third spikes of SlHZ41 transgenic plants and wild plants 3 months after transplantation; Figure 8 Figure D in Figure 8 is the fruit peel thickness of the second and third spikes of SlHZ41 transgenic plants and wild plants 3 months after transplantation; Figure 8 Figure E in Figure 8 is the single fruit weight of the second and third spikes of SlHZ41 transgenic plants and wild plants 3 months after transplantation.

[0033] Figure 9 In the present invention is the expression profile of SlHDZ I subfamily members under heat stress. Detailed implementation mode

[0034] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well-known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0035] Example 1

[0036] I. Obtaining of the SlHZ41 gene

[0037] According to the analysis of the expression patterns of HD-Zip I subfamily members under heat treatment based on the previous laboratory transcriptome data, it was shown that SlHZ41 was significantly induced by high temperature. The analysis results are as Figure 9 shown.

[0038] II. Tomato High Temperature Treatment

[0039] High temperature stress: Select plump tomato 'Ailsa Craig' AC seeds, disinfect them and inoculate them on a 1 / 2MS solid medium plate of 20 cm × 20 cm. After culturing in an artificial climate chamber at 25 °C, relative humidity of 70%, and light intensity of about 800 μmol m- 2 s -1 for 12 days, transfer the plate to a light incubator at 42 °C, relative humidity of 70%, and light intensity of about 800 μmol m -2 s -1 for high temperature stress treatment. Select tomato seedlings at 0 h, 2 h, 4 h, 6 h, 12 h, 24 h, 48 h after high temperature treatment and those after 24 h of high temperature treatment and then recovered in a 25 °C light incubator for 1 day for sampling.

[0040] Among them, the tomato 'Ailsa Craig' AC seeds were from the team of Professor Zhan Xiangqiang of the Laboratory of Horticultural Crop Stress Biology, Northwest A&F University.

[0041] The formula of 1 / 2MS medium is: Weigh 2.23 g of MS powder, dissolve it in 800 mL of ultrapure water, add 15 g of sucrose, and make up the volume to 1000 mL. Adjust the PH to 5.8, add 7.5 g of agar, and autoclave at 121 °C for 21 min. Among them, MS powder: Phytotech, M519.

[0042] In 2022, the following tissue samples were obtained from tomato plants in the fruiting stage in the artificial climate chamber: Rt: root; St: stem; YL: young leaf; OL: old leaf; LB: lateral branch; FL: flower; MG: green mature fruit; B: breaker fruit; B+3: breaker + 3 days fruit; B+7: breaker + 7 days fruit; B+10: breaker + 10 days fruit; B+15: breaker + 15 days fruit; seed: seed.

[0043] Among them, "young leaf" refers to the leaves at the first and second node positions counted from the top to the bottom of the plant.

[0044] "Old leaf" refers to the leaves at the fourth and fifth node positions counted from the top to the bottom of the plant.

[0045] "Green mature" means that the size of the tomato fruit has grown to the maximum and no longer swells.

[0046] "Color break" refers to the stage when tomato fruits show slightly yellow-orange color and begin to turn towards maturity. Total RNA of tomatoes was extracted using the NGzol Total RNA Rapid Extraction Kit from Shanghai Huiling Co., Ltd. according to the kit instructions and stored at -80 °C for later use. Reverse transcription was carried out using the HiScript II 1st Strand cDNA Synthesis Kit: +gDNAWiper from Novoprotein Co., Ltd., and the quantitative kit ChamQ SYBR qPCR Master Mix was purchased from Novoprotein Co., Ltd. The relative expression levels of genes were detected using the CFX96 Real-Time PCR Detection System from Bio-Rad Co., Ltd. Among them, the quantitative kit ChamQ SYBR qPCR Master Mix was purchased from Novoprotein Co., Ltd., cat.no.Q311-01.

[0047] The expression of SlHZ41 in different tissues of AC: roots, stems, new leaves, old leaves, lateral branches, flowers, green-ripe fruits, color-break fruits, color-break + 3 days fruits, color-break + 7 days fruits, color-break + 10 days fruits, and seeds was determined by qRT-PCR. The results are as Figure 1 shown.

[0048] The results showed that the SlHZ41 gene was expressed in all tissues, with the highest expression level at 7 days after color break of fruits, followed by seeds, flowers, old leaves, stems, and roots.

[0049] The expression of SlHZ41 under high temperature stress treatment at 42 °C for 0 h, 2 h, 4 h, 6 h, 12 h, 24 h, 48 h and after recovery was determined by qRT-PCR. The results are as Figure 2 shown. The results showed that the SlHZ41 gene was significantly heat-induced during 2 h - 4 h of high temperature treatment at 42 °C, suggesting that the SlHZ41 gene might be involved in the high temperature stress response process of tomato plants.

[0050] III. Construction of knockout vector

[0051] 3.1 Primer design for sgRNA sequence

[0052] The single-stranded guide RNA sequence of the tomato SlHZ41 gene was selected using the CRISPR-PLANT website. sgRNA, as the target guiding sequence for CRISPR-Cas9 knockout, was used to design the oligonucleotide sequences of SlHZ41-SgRNA-F and SlHZ41-SgRNA-R.

[0053] The nucleotide sequence of sgRNA is shown in SEQ ID NO.16: CAGAGTCAAGGCCTGAATTG.

[0054] The nucleotide sequences of SlHZ41-SgRNA-F and SlHZ41-SgRNA-R are as follows:

[0055] The nucleotide sequence of SlHZ41-SgRNA-F is shown in SEQ ID NO.4: gattgCAGAGTCAAGGCCTGAATTG;

[0056] The nucleotide sequence of SlHZ41-SgRNA-R is shown in SEQ ID NO.5: aaacCAATTCAGGCCTTGACTCTGc.

[0057] Among them, the CRISPR-PLANT website is http: / / www.genome.arizona.edu / crispr / .

[0058] 3.2 Construction of CRISPR-Cas9 knockout vector

[0059] (1) Linearize the PSgR-Cas9-At vector. Use BbsI to digest the vector overnight at room temperature to linearize it. After electrophoresis detection, purify and recover it. The detection results are shown in Figure 3 Figure A in it.

[0060] Among them, the digestion system: 0.25 μL of Bbs I, 1 μL of CIP, 3 μL of psgR-Cas9-At vector, 1.12 μL of NEB buffer, 3.75 μL of ddH2O.

[0061] "Overnight" means the time ≥ 12 h.

[0062] (2) Phosphorylate the sgRNA. Perform the following reaction in a PCR instrument to obtain phosphorylated sgRNA.

[0063] Among them, the reaction system: 0.5 μL of T4 PNK, 1 μL of SlHZ41-SgRNA-F, 1 μL of SlHZ41-SgRNA-R, 1 μL of 10×T4 Ligation buffer, 6.5 μL of ddH2O. The reaction program: 37°C for 30 min, 95°C for 5 min, with a temperature gradient decrease of 5°C per minute until the temperature drops to 25°C and then ends.

[0064] The phosphorylated sgRNA was ligated to the linearized PSgR-Cas9-At vector. Reaction system: 2 μL of BbsI digested psgRCas9-At, 1 μL of phosphorylated sgRNA diluted 200-fold, 1 μL of 10× T4 Ligation buffer, 0.25 μL of T4 Ligase, 5.75 μL of ddH2O. After 1 h of ligation reaction at room temperature, Escherichia coli was transformed by heat shock method. The transformed cells were spread on a solid LB plate containing 100 mg / L ampicillin antibiotic. PCR detection was performed using M13F and SlHZ41-SgRNA-R. The detection results were as shown in Figure B of Figure 3 , and the SlHZ41-SgRNA-Cas9 intermediate vector was obtained. The recombinant plasmid of the positive clone was extracted according to the operation steps of the plasmid extraction kit of Shanghai Huiling Company to obtain the SlHZ41-SgRNA-Cas9 intermediate vector.

[0065] Among them, the nucleotide sequence of M13F is as shown in SEQ ID NO.8: 5'-TGTAAAACGACGGCCAGT-3'.

[0066] Method for transforming Escherichia coli by heat shock method:

[0067] S1. Take out the prepared competent cells from the -80°C refrigerator and place them on ice to slowly dissolve.

[0068] S2. Add 5 μL of plasmid and gently mix well.

[0069] S3. Incubate on ice for 30 min, then heat shock at 42°C for 90 s, and then immediately place on ice for 2 min.

[0070] S4. Perform the operation in a laminar flow hood and add 700 μL of LB culture medium.

[0071] S5. Incubate on a shaker at 37°C for 60 min.

[0072] S6. After the resuscitation is completed, centrifuge at 8000 rpm for 3 min.

[0073] S7. Remove part of the supernatant, gently suspend the remaining precipitate, and spread it on an LB plate containing antibiotics.

[0074] S8. Incubate inverted in a 37°C incubator for 12 h.

[0075] (3) Digest the SlHZ41-SgRNA-Cas9 PCR intermediate vector and the pCAMBIA2301 vector. The reaction is as follows: 10 μL of pCAMBIA2301 / SlHZ41-SgRNA-Cas9 plasmid, 3 μL of 10×Cutsmart buffer, 1 μL of EcoR I-HF, 1 μL of Hind III-HF, and 15 μL of ddH2O. After mixing, react at 37°C for 2 h, and purify and recover the target fragment (the SlHZ41-SgRNA-Cas9 fragment is as shown in Figure C of Figure 3 and the linearized pCAMBIA2301 vector is as shown in Figure D of Figure 3 .

[0076] Ligate the sgRNA-Cas9 fragment and the linearized pCAMBIA2301 vector. The reaction system is as follows: 2 μL of linearized pCAMBIA2301 vector, 4 μL of linearized SlHZ41-SgRNA-Cas9 fragment, 1 μL of 10×T4 Ligation buffer, 0.2 μL of T4 Ligase, and 2.8 μL of ddH2O. After the ligation reaction at room temperature for 2 h, transform the Escherichia coli competent cells by heat shock method, and perform colony PCR detection on the solid LB medium containing 50 mg / L Kana. The detection primers for colony PCR detection are pCAMBIA2301-F and SlHZ41-SgRNA-R; the nucleotide sequence of pCAMBIA2301-F is as shown in SEQ ID NO.9: 5'-CGGCGAGTTCTGTTAGGTCCTCTA-3'. Screen the positive clones and extract the plasmids. The result is as shown in Figure 3E, and SlHZ41-Cas9-pCAMBIA2301 is obtained.

[0077] IV. Construction of the overexpression vector of the SlHZ41 gene

[0078] Download the CDS sequence of the SlHZ41:Solyc08g083130 gene from the tomato database SGN, design the specific primers OE-SlHZ41-F and OE-SlHZ41-R for the gene, and PCR amplify the full-length cDNA sequence of the SlHZ41 gene.

[0079] Among them, the website of the tomato database SGN is https: / / solgenomics.net / .

[0080] The 25 μL PCR amplification system is as follows: 12.5 μL of 2× High MasterMix, 1 μL of OE-SlHZ41-F, 1 μL of OE-SlHZ41-R, 1 μL of template cDNA, and 9.5 μL of ddH2O. Pre-denaturation at 98°C for 3 min; 98°C for 10 s, 57°C for 30 s, 72°C for 40 s, for 35 cycles; 72°C for 5 min.

[0081] Finally, purify and recover the cloned gene fragment. The recovered SlHZ41 gene fragment is as shown in Figure 4 Figure A in

[0082] Construct an overexpression vector:

[0083] (1) Linearize the pHellsgate2 vector with Xho I and Xba I restriction endonucleases. The linearized pHellsgate2 vector is as shown in Figure 4 Figure B in

[0084] (2) Ligate the target gene fragment: the SlHZ41 gene fragment to the linearized vector. Use the ClonExpress II One Step Cloning Kit homologous recombination reaction system for ligation as follows: 4 μL of the SlHZ41 gene fragment, 2 μL of 5× CeII buffer, 1 μL of 5× CeII Ligase, 2 μL of the linearized pHellsgate2 Vector, and 1 μL of H2O. The reaction condition is to react at 37°C for 30 min in a PCR instrument. The ligation product is transformed into Escherichia coli competent cells by heat shock method. After colony PCR detection, identify positive clones and extract plasmids. The result is as shown in Figure 4 Figure C in

[0085] Among them, ClonExpress II One Step Cloning Kit: Vazyme, nanjing, China.

[0086] The detection primers for colony PCR detection are 35S-F and OE SlHZ41-R.

[0087] The nucleotide sequence of 35S-F is as shown in SEQ ID NO.10: 5'-GACGCACAATCCCACT ATCC-3'; The nucleotide sequence of OESlHZ41-R is as shown in SEQ ID NO.11: 5'-TCTCATT AAAGCAGGACTCTAGACTAAAATTCCCACAATGGTGATG-3'.

[0088] The operation steps for transforming Escherichia coli competent cells by heat shock method are the same as above.

[0089] (3) Send the positive plasmid to Beijing Tsingke Biotechnology Co., Ltd. for sequencing detection.

[0090] The nucleotide sequence of the SlHZ41 gene measured is shown as SEQ ID NO.1 as follows:

[0091] ATGGCTCCAACAAACTCAGACATGATTTGGGGTGAATCAATCATGTCAAGTAAGTGCAACGACAACATGAGAAGAAGGTTCAATGATGAGCAGATTAAGTCATTAGAAAACATGTTTGAGACAGAGTCAAGGCCTGAATTGAGGACAAAACAGCAACTGGCTAAAAGGCTTGGACTGCAGCCAAGACAAGTAGCTATATGGTTTCAGAACAAGAGAGCTAGATCGAAATCGAAGCAACTTGAATTGGAGTATAGAATGCTTCAAATCAGTTATGACAACTTAGGTTCCAAGTATGAATTACTCAAAAAAGAGCATGAATCCCTCCTCATCCAGCTGCAGAGACTTAAAAAGTTAATGGAAAAGGATGACAATGAGAAAGATGTGAATAAAAAATCAGAAACGGAAGTGAAACAAGATGATTTTGCACCAGAATTTGGCAGCAGGGGAATTGACTACTTGAGAGCAGAATCTGATATTTTAGACATGGCACAAATAGCTGATGGCTTATCGGAGATTGAAAATGAATTCAACTTCGAGTCCAGAACGTTTCTTCATGATACTGGTTGTACATCACCATTGTGGGAATTTTAG.

[0092] The amino acid sequence of the protein expressed by the above SlHZ41 gene is shown as SEQ ID NO.2 as follows:

[0093] MAPTNSDMIWGESIMSSKCNDNMRRRFNDEQIKSLENMFETESRPELRTKQQLAKRLGLQPRQVAIWFQNKRARSKSKQLELEYRMLQISYDNLGSKYELLKKEHESLLIQLQRLKKLMEKDDNEKDVNKKSETEVKQDDFAPEFGSRGIDYLRAESDILDMAQIADGLSEIENEFNFESRTFLHDTGCTSPLWEF*。

[0094] The sequence of the target site of the above-mentioned SlHZ41 gene is shown in SEQ ID NO.3:

[0095] CAGAGTCAAGGCCTGAATTG。

[0096] V. Agrobacterium-mediated genetic transformation of tomato

[0097] 5.1 Tomato seed treatment

[0098] On the ultra-clean workbench, soak tomato 'Ailsa Craig' AC seeds in sterile water for 30 min. After the tomato seeds have imbibed water, pour out the clear water, add 75% (v / v) alcohol to the Erlenmeyer flask, shake for 30 s and then pour out the alcohol. Then wash the seeds with the disinfectant solution for 10 min, shaking constantly during this period. Wash the tomato seeds 3 times with sterile ddH2O and then inoculate the tomato seeds on 1 / 2 MS medium, and culture them in an artificial climate chamber at 25 °C with a 16 h / 8 h light cycle for 7 d. At this time, the cotyledons of most seedlings are fully expanded.

[0099] Among them, the disinfectant solution is 84 disinfectant: distilled water = 1:1.

[0100] 5.2 Preparation of explants

[0101] On the ultra-clean workbench, select sterile seedlings with fully expanded cotyledons, cut each cotyledon into 2 explants with a scalpel, and place the obtained tomato cotyledon nodes on the pre-culture medium, and culture them in the dark at 25 °C for 1 d to obtain tomato explants.

[0102] Among them, the pre-culture medium is KCMS medium, and its formula is: weigh 4.43 g / L MS powder, 30 g / L sucrose, adjust the pH to 5.8, 7.5 g / L agar, and autoclave at 121 °C for 21 min. Among them, the MS powder: Phytotech, M524.

[0103] 5.3 Agrobacterium infection and transformation of tomato

[0104] (1) Streak-activate the GV3101 Agrobacterium containing pHellsgate2:SlHZ41 or SlHZ41-Cas9-pCAMBIA2301 on an LB solid medium containing 50 mg / L Rif and 50 mg / L Kan.

[0105] (2) Pick a monoclonal colony and add it to 15 mL of LB liquid medium containing 50 mg / L Rif and 50 mg / L Kan, and culture it with shaking at 28 °C until the OD 600 reaches about 0.8.

[0106] (3) Centrifuge to collect the bacteria, add an equal volume of Agrobacterium suspension, and simultaneously add 100 mmol / L AS to prepare an Agrobacterium infection solution.

[0107] (4) Place the obtained tomato explants into an empty glass dish, pour in the Agrobacterium infection solution, shake continuously for 10 min during this period, and finally transfer the tomato explants to the pre-culture medium and continue dark culture for 2 d.

[0108] Among them, the LB liquid medium: yeast extract 5 g / L, tryptone 5 g / L, sodium chloride 10 g / L; LB solid medium.

[0109] Yeast extract 5 g / L, tryptone 5 g / L, sodium chloride 10 g / L and agar 15 g / L.

[0110] Agrobacterium suspension: Weigh 4.43 g / L of MS powder, 30 g / L of sucrose, adjust the pH to 5.8, and autoclave at 121 °C for 21 min. Among them, the MS powder: Phytotech, M524.

[0111] 5.4 Screening culture

[0112] Transfer the tomato explants co-cultured with Agrobacterium for 2 d to the screening medium, place about 30 explants in each culture dish with the leaf surface facing up, and culture them in an incubator at 25 °C with a 16 h / 8 h light cycle for about 15 d until adventitious buds regenerate from the callus.

[0113] Among them, the screening medium: 4.43 g / L of MS powder, 30 g / L of sucrose, 7.4 g / L of agar, 0.1 mg / L of IAA, 2 mg / L of zeatin ZR, 100 mg / L of kanamycin Kana and 360 mg / L of ticarcillin, pH 5.8. Among them, the MS powder: Phytotech, M519.

[0114] 5.5 Subculture

[0115] The selected tomato explants were transferred back to the subculture medium and cultured in a phytotron at 25°C with a 16h / 8h light / dark cycle for about 15 days until adventitious buds regenerated from the callus.

[0116] Among them, the subculture medium: 4.43 g / L MS powder, 30 g / L sucrose, 7.4 g / L agar, 0.2 mg / L zeatin riboside (ZR), 100 mg / L kanamycin (Kana), and 360 mg / L ticarcillin (TMT), pH 5.8. Among them, the MS powder: Phytotech, M519.

[0117] 5.6 Rooting culture

[0118] When the adventitious buds grew to 2 cm, they were cut from the base and transferred to the rooting medium to induce root formation.

[0119] Among them, the rooting medium: 4.43 g / L MS powder, 30 g / L sucrose, 7.4 g / L agar, 2 mg / L indole-3-butyric acid (IBA), pH 5.8, 360 mg / L TMT + 50 mg / L Kana. Among them, the MS powder: Phytotech, M519.

[0120] 5.7 Transplanting and acclimatization

[0121] When the adventitious roots of the tomato seedlings grew well, the tomato seedlings were taken out, the root medium was washed, and they were transplanted into a culture pot filled with vermiculite. A transparent lid was put on to keep the humidity for about one week, and then the transparent lid was removed for acclimatization for 2 weeks. Finally, the transgenic tomato plants could be transplanted into the greenhouse for normal field management for seed propagation.

[0122] Among them, the normal field management is as follows: Do a good job in plant arrangement, and timely carry out field management such as pruning, pinching, tying vines, and removing old leaves to facilitate ventilation and light transmission.

[0123] VI. Detection of transgenic plants

[0124] 6.1 Identification of overexpression plants

[0125] DNA of transgenic plants and wild-type tomato plants was extracted by the CTAB method. The specific operation is as follows:

[0126] (1) Take fresh leaves and place them in a 2 mL EP tube containing steel beads. After quick freezing in liquid nitrogen, grind them into powder on a grinder.

[0127] (2) Quickly add 1 mL of CTAB extraction buffer, shake well, and incubate in a 65°C water bath for 15 min.

[0128] (3) Add 500 μL of chloroform, shake vigorously, and then centrifuge at 12000 rpm for 10 min.

[0129] (4) Carefully pipette 400 μL of the supernatant, add an equal volume of isopropanol, mix well by shaking, and let it stand for about 30 min for precipitation reaction in a -20 °C refrigerator.

[0130] (5) Centrifuge at 12000 rpm for 10 min and discard the supernatant.

[0131] (6) Add 750 μL of 75% (v / v) ethanol, mix well by shaking, centrifuge at 12000 rpm for 5 min, discard the supernatant, and place it in a fume hood to completely evaporate the ethanol.

[0132] (7) Finally, add 100 μL of 10 mM, pH 8.0 Tris-HCl solution, mix well, and store the DNA sample in a -20 °C refrigerator.

[0133] Perform PCR detection on the DNA of the transgenic plants and wild-type tomato plant leaves obtained by the above extraction.

[0134] Among them, the PCR reaction system is: 10 μL of 2×Taq Master Mix, 0.5 μL of 35S-F (10 μM), 0.5 μL of OESlHZ41-R (10 μM), 2 μL of DNA, 7 μL of ddH2O.

[0135] The PCR reaction program is: ① Pre-denaturation, react at 94 °C for 5 min; ② Denaturation, react at 94 °C for 30 s; ③ Annealing, react at 57 °C for 30 s; ④ Extension, react at 72 °C for 1 min; ⑤ Post-extension, react at 72 °C for 5 min, and cycles ②③④ are repeated 33 times.

[0136] The PCR reaction primers are 35S-F and OE SlHZ41-R.

[0137] The nucleotide sequence of 35S-F is shown in SEQ ID NO.10; 5'-GACGCACAATCCCACT ATCC-3'; the nucleotide sequence of OESlHZ41-R is shown in SEQ ID NO.11; 5'-TCTCATTA AAGCAGGACTCTAGACTAAAATTCCCACAATGGTGATG-3'.

[0138] According to the PCR detection results of the above DNA, DNA-positive plants were selected for RNA-level detection. The specific operation was as follows: The total RNA of tomatoes was extracted using the NGzol Total RNA Rapid Extraction Kit from Shanghai Huiling Co., Ltd. according to the kit instructions. Reverse transcription was performed using the HiScript II 1st Strand cDNA Synthesis Kit: +gDNAWiper from Novoprotein Co., Ltd. The quantitative kit ChamQ SYBR qPCR Master Mix was purchased from Novoprotein Co., Ltd., and the relative gene expression was detected using the CFX96 fluorescence quantitative PCR instrument from Bio-Rad Co., Ltd. Among them, the quantitative kit ChamQ SYBR qPCR Master Mix was purchased from Novoprotein Co., Ltd., cat.no.Q311-01.

[0139] The qRT-PCR primers were SlHZ41-qPCR-F and SlHZ41-qPCR-R.

[0140] The nucleotide sequence of SlHZ41-qPCR-F was as shown in SEQ ID NO.12: 5'-GAAGAAGGTTCAATGATGAGCAG-3'; the nucleotide sequence of SlHZ41-qPCR-R was as shown in SEQ ID NO.13: 5'-AGTCTCTGCAGCTGGATGAGG-3'.

[0141] The qRT-PCR system: 2×ChamQ Universal SYBR qPCR Master Mix 7.5 μL, 0.3 μL SlHZ41-qPCR-F (10 μM), 0.3 μL SlHZ41-qPCR-R (10 μM), 2 μL Template DNA / cDNA, 4.9 μL ddH2O,

[0142] The qRT-PCR program: Pre-denaturation: 95°C, 5 min; 40 cycles: Denaturation: 95°C, 10 s; Annealing: 60°C, 30 s; Extension 72°C, 30 s to collect fluorescence. Using tomato Actin7 as the internal reference gene, each sample had 3 technical replicates, -ΔΔCt Calculate the relative expression level.

[0143] The nucleotide sequence of Actin7-RT-F was as shown in SEQ ID NO.14: 5'-AGAAGGATGCGTATGTGGGT-3';

[0144] The nucleotide sequence of Actin7-RT-R was as shown in SEQ ID NO.15: 5'-AGAAGGATGCGTATGTGGGT-3'.

[0145] 6.2 Identification of CRISPR / Cas knockout plants

[0146] DNA of transgenic plants and wild - type tomato plant leaves was extracted by the CTAB method. pCAMBIA2301 - F and SlHZ41 - SgRNA - R were used for the detection of positive seedlings. DNA sequencing primers: SlHZ41 - check - F and SlHZ41 - check - R were designed about 200 bp before and after the sgRNA sequence of the target site of the SlHZ41 gene using Primer5. The DNA of positive plants detected by DNA was used as a template to amplify the fragment containing the sgRNA target site, and the PCR product was sequenced and compared with the reference sequence to determine whether mutations occurred and the mutation sites.

[0147] Among them, the nucleotide sequence of SlHZ41 - check - F is shown in SEQ ID NO.6: 5'-ATGGCTCCAACAAACTCAGACAT-3'; the nucleotide sequence of SlHZ41 - check - R is shown in SEQ ID NO.7: 5'-ATTTCGATCTAGCTCTCTTGTTCTG-3'.

[0148] VII. Heat tolerance identification

[0149] Tomato AC seeds with plump grains, two SlHZ41 over - expression lines with higher expression levels and tomato seeds of the SlHZ41 CRISPR / Cas homozygous knockout line were selected for germination in a constant temperature incubator at 28°C. The germinated seeds were sown in nutrient pots and cultured in an artificial climate chamber with 16 h light / 8 h dark, 24°C / 20°C. When the plants grew to four leaves and one heart, the wild - type WT, mutant plants and over - expression plants were divided into two groups, one group as the control group and the other group as the experimental group. The experimental group was subjected to high - temperature stress treatment.

[0150] Among them, the nutrient pot is a mixed substrate obtained by mixing peat: vermiculite: perlite with a volume ratio of 3:1:1.

[0151] The method of high - temperature treatment is as follows: Tomato materials of different lines were randomly placed in an artificial climate chamber at 42°C, with 15 plants of each line treated. During the treatment, the air humidity in the climate chamber was maintained at about 50%, ensuring sufficient water in the roots of tomato plants to avoid causing drought stress. The plant phenotypes were recorded, and at the same time, leaf materials at the same node of plants of different lines were selected for the determination of related physiological and biochemical indexes; SOD activity, POD activity, malondialdehyde content and relative conductivity.

[0152] Tomato leaves before and after treatment were weighed and then quickly frozen in liquid nitrogen. They were ground with a high - throughput sample grinder and reserved.

[0153] Enzyme solution extraction: Add 1 mL of phosphate buffer and mix thoroughly by shaking. Centrifuge at 13,000 g for 20 min at 4 °C. The supernatant is the enzyme solution. Pipette the supernatant into a centrifuge tube and place it on ice for later use.

[0154] 7.1 Determination of SOD activity

[0155] Add 50 mM PBS (pH = 7.8), 130 mM methionine, 750 μM nitroblue tetrazolium solution, 100 μM EDTA-Na2, 20 μM riboflavin solution, and the enzyme solution into a test tube in sequence. At the same time, set up a light control and a dark control, and measure the absorbance of the solution at a wavelength of 560 nm.

[0156] SOD activity (unit / g FW) = ((Absorbance of the control tube - Absorbance of the sample tube) * Volume of the extraction solution) / (Absorbance of the control tube * 0.5 * Fresh weight of the sample * Volume of the enzyme solution used in the determination) = ((ACK - AE) * 0.8) / (ACK * 0.5 * FW * 0.1).

[0157] Where, ACK: Absorbance of the control tube; AE: Absorbance of the sample tube; 0.5: Enzyme activity coefficient at half unit.

[0158] The results are shown in Figure 7 . Figure 7 Figure (A) in shows the phenotype after high-temperature treatment; Figure 7 Figure (B) in shows DAB staining; Figure 7 Figure (C) in shows SOD activity; Figure 7 Figure (D) in shows relative conductivity; Figure 7 Figure (E) in shows malondialdehyde content.

[0159] 7.2 Determination of POD activity

[0160] Add 50 mM PBS, 100 mM H2O2, 100 mM guaiacol, and the enzyme solution into a test tube in sequence. At a wavelength of 470 nm, measure the absorbance of the solution using a kinetic curve.

[0161] POD activity (μmol g-1 s-1) = (ΔA470 * 0.8) / (0.015 * FW * 26.8 * 60 * 3) = (A470 * Volume of the extraction solution) / (Volume of the enzyme solution used in the determination * Fresh weight of the sample * Extinction coefficient * Time).

[0162] Where, the extinction coefficient is 26.8 and the time is expressed in minutes.

[0163] The pH of PBS is 7.0.

[0164] The results are shown in Figure 7 .

[0165] 7.3 Determination of malondialdehyde content

[0166] Add enzyme solution and 0.5% (v / v) thiobarbituric acid into a finger tube, mix well, place in a boiling water bath for 20 min, quickly cool to room temperature on ice, centrifuge at 3000 rpm for 10 min at 4 °C, and aspirate the supernatant to measure the absorbance of the enzyme solution at wavelengths of 532 nm, 600 nm and 450 nm.

[0167] MDA concentration (μmol / L) = 6.45 * (A532 - A600) - 0.56 * A450.

[0168] MDA content (μmol / g) = MDA concentration (μmol / L) * extraction volume (mL) / fresh weight of plant tissue (g).

[0169] The results are shown in Figure 7 .

[0170] 7.4 Determination of relative electrical conductivity

[0171] Use a tool to punch ten leaf discs and place them in a glass tube containing 10 mL of deionized water. Oscillate at 100 times per minute for 2 h under normal temperature conditions, and measure the initial electrical conductivity (C1) with a conductivity meter. Then, boil the glass tube containing the leaf discs in distilled water for 30 minutes, and measure the final electrical conductivity (C2) after cooling the tube to ambient temperature. Measure the electrical conductivity of deionized water (C0) and use it as a blank. REC is calculated as (C1 - C0) / (C2 - C0) × 100%.

[0172] The results are shown in Figure 7 .

[0173] Through phenotypic observation, it was found that after 24 h of high-temperature treatment, the top of the wild-type plants showed wilting, the overexpressing plants wilted and dried up as a whole, and the knockout plants grew well. H2O2 is a major reactive oxygen species (ROS) molecule, which usually accumulates under abiotic stress. The DAB staining (detecting H2O2) method was used to detect the accumulation of O2 and H2O2 in the AC, overexpressing plants and knockout plants under control and high-temperature conditions. Under control conditions, there was no difference in the accumulation of H2O2 among all plants. Under high-temperature treatment, the accumulation of O2 and H2O2 in the overexpressing plants was the highest, the accumulation of H2O2 in the knockout plants was the lowest, and the wild-type plants were between the two.

[0174] The detection results of SOD and POD activities showed that the activities of SOD and POD in the knockout plants were significantly higher than those in the wild-type plants, while the activities of SOD and POD in the overexpressing plants were significantly lower than those in the wild-type plants.

[0175] The content of malondialdehyde represents the degree of membrane damage. After heat treatment, the content of malondialdehyde in overexpressing plants is the highest, indicating that the degree of damage to overexpressing plants is the most obvious after heat treatment.

[0176] The above results indicate that the SlHZ41 gene negatively regulates tomato heat resistance.

[0177] VIII. Field trait statistics

[0178] Tomato plants were planted in a glass greenhouse and managed routinely. The size and single fruit weight of the fruits in the second and third trusses were investigated.

[0179] Among them, for routine management, plant tidying was done well, and field management such as pruning, pinching, tying vines, and removing old leaves was carried out in a timely manner to facilitate ventilation and light transmission. The results are shown in Figure 8 . Figure 8 Figure A in shows the fruit phenotypes of the second and third trusses of SlHZ41 transgenic plants and wild plants 3 months after planting; Figure 8 Figure B in shows the phenotypes of the whole plants of SlHZ41 transgenic plants and wild plants 3 months after planting; Figure 8 Figure C in shows the fruit quality of the second and third trusses of SlHZ41 transgenic plants and wild plants 3 months after planting; Figure 8 Figure D in shows the fruit peel thickness of the second and third trusses of SlHZ41 transgenic plants and wild plants 3 months after planting; Figure 8 Figure E in shows the single fruit weight of the second and third trusses of SlHZ41 transgenic plants and wild plants 3 months after planting.

[0180] By observing the single fruit weight, fruit peel thickness and yield of transgenic plants, it was found that overexpression of the SlHZ41 gene reduced the tomato yield, and knocking out had the opposite effect.

[0181] In summary, it is shown that by statistically analyzing the fruit size, yield and overexpression of SlHZ41, the tomato yield is reduced, while knocking out SlHZ41 increases the tomato yield.

[0182] It should be noted that when the present invention involves numerical ranges, it should be understood that any value between the two endpoints of each numerical range and the two endpoints can be selected. To prevent repetition, preferred embodiments of the present invention are described.

[0183] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept, and these changes and modifications all fall within the scope of all changes and modifications of the present invention.

Claims

1. SlHZ41 Use of a gene in improving heat tolerance and fruit size of tomatoes, characterized in that, Knock out the SlHZ41 gene to improve the heat tolerance and fruit size of tomatoes, and the SlHZ41 nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the amino acid sequence of the protein expressed by it is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that By means of genetic engineering, knockout the said SlHZ41 gene, or reduce the SlHZ41 expression level of the gene, thereby obtaining a tomato mutant with improved heat tolerance and fruit size.

3. A method for improving the heat tolerance and fruit size of tomatoes, characterized in that, Use the CRISPR / Cas9 gene editing technology to site-specifically edit the target site of the gene as described in claim 1 in tomatoes SlHZ41 to knockout the said SlHZ41 gene; The SlHZ41 nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the amino acid sequence of the protein expressed thereby is shown in SEQ ID NO. 2; The sequence of the target site of the SlHZ41 gene is shown as SEQ ID NO.

3.

4. The method according to claim 3, characterized in that, Link the sgRNA targeting the target site of the SlHZ41 gene into a transgenic vector to construct a recombinant expression vector; Transfect the recombinant expression vector into the plant material to be transformed to obtain SlHZ41 gene-edited site-directed knockout tomato plants, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO.

16.

5. The method according to claim 4, characterized in that, The transgenic vector is pCAMBIA2301.

6. A method for cultivating heat-resistant and fruit size-improved tomatoes, characterized in that, It includes the following steps: Based on the nucleotide sequence of the gene as described in claim 1 SlHZ41 determine the sequence of the target site, synthesize sgRNA primers for the target site, the sequence of the target site is as shown in SEQ ID NO. 3, the sgRNA primer sequences are SlHZ41-SgRNA-F and SlHZ41-SgRNA-R, the nucleotide sequence of SlHZ41-SgRNA-F is as shown in SEQ ID NO. 4, and SlHZ41-SgRNA-R is as shown in SEQ ID NO. 5; Performing PCR amplification using the sgRNA primer to obtain phosphorylated sgRNA; Construct a recombinant expression vector containing the phosphorylated sgRNA to knock out the SlHZ41 gene in tomato; Transferring the recombinant expression vector into Agrobacterium to obtain recombinant Agrobacterium; Transfecting the recombinant Agrobacterium into the tomato to be transformed, and through selection, differentiation, rooting, acclimatization, and transplantation, obtaining heat-resistant and fruit size-improved tomato plants.

7. The cultivation method according to claim 6, characterized in that, After transfecting the recombinant Agrobacterium into the tomato to be transformed, tomatoes are obtained through tissue culture SlHZ41 Positive transgenic seedlings, which are confirmed by PCR amplification and sequencing SlHZ41 Tomato plants with gene knockout are obtained.

8. The cultivation method according to claim 7, characterized in that, The primers used for the PCR amplification are SlHZ41-check-F and SlHZ41-check-R; Among them, the nucleotide sequence of SlHZ41-check-F is shown as SEQ ID NO. 6, and the nucleotide sequence of SlHZ41-check-R is shown as SEQ ID NO. 7.

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