SlHZ41 gene and application thereof in improving heat resistance and fruit size of tomato

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to an SlHZ41 gene and application thereof in improving heat resistance and fruit size of tomatoes. The nucleotide sequence of the SlHZ41 gene provided by the invention is as shown in SEQ ID NO. 1, and the amino acid sequence of the protein expressed by the SlHZ41 gene is as shown in SEQ ID NO. 2. The SlHZ41 gene knockout vector provided by the invention can be used for improving POD and SOD activity of plants, inhibiting accumulation of MDA content and reducing damage of high temperature stress to the plants; meanwhile, the yield of tomatoes is increased by knocking out plants; a theoretical basis is provided for discussing the resistance mechanism, the field yield and the gene utilization of the SlHZ41 gene.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a SlHZ41 gene and an application thereof in improving heat resistance and fruit size of tomatoes. Background Art

[0002] Tomato (Solanum lycopersicum L.) is an important vegetable crop widely cultivated around the world. It is rich in vitamin C, lycopene, dietary fiber, potassium, β-carotene, etc. It has antioxidant, heart health protection, cancer prevention, vision protection, and anti-inflammatory effects. According to statistics from the Food and Agriculture Organization of the United Nations (FAOSTAT), my country's tomato planting area reached 1.137 million hectares in 2022, with a production of 68.24 million tons, close to 1 / 3 of the world's production.

[0003] Tomatoes are sensitive to environmental temperature. Domestic cultivation is mainly carried out in simple greenhouses and open fields. The management is relatively extensive and they are easily harmed by various adverse stresses such as high temperature. Among them, high temperature has become one of the important limiting factors for tomato production, often causing huge economic losses. In order to ensure the stable and sustainable development of the tomato industry, it is necessary to give priority to breeding, use 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 commodity quality, it is also necessary to accelerate the selection of high temperature and other adverse conditions resistant varieties.

[0004] The homeodomain-leucine zipper (HD-Zip) transcription factor family is a class of transcription factors unique to plants, and is widely involved in the response process of plant growth and development and stress response. Family members generally have the characteristics of being induced by stress and participate in the regulation of hormone signals and various stress responses. The expression of Arabidopsis AtHB5, AtHB6, AtHB7, and AtHB12 is induced by drought and abscisic acid treatment. Overexpression of alfalfa MsHB7 reduces the salt stress tolerance of transgenic plants. Tomato SlHB15A can bind to the SlIAA32 promoter to regulate auxin signals and thus regulate tomato pedicel abscission. Tomato HD-ZipⅣ subfamily member 8 is involved in the development of tomato epidermal hairs. However, the role of tomato HD-Zip in the regulation of high temperature tolerance and fruit size has been rarely reported. Summary of the invention

[0005] In order to solve the problem that there is little report on the role of tomato HD-Zip in regulating high temperature tolerance and fruit size in the prior art, the present invention adopts the following technical solution:

[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 the 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] The SlHZ41-Cas9-pCAMBIA2301-containing vector is transferred into Agrobacterium to obtain recombinant Agrobacterium; the recombinant Agrobacterium is transfected into tomatoes to be transformed, and heat-resistant and fruit-size improved tomato plants are obtained through selection, differentiation, rooting, seedling hardening and transplanting.

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

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

[0019] Preferably, after the recombinant Agrobacterium is transfected into the tomato to be transformed, tomato S1HZ41 transgenic positive seedlings are obtained by tissue culture, the S1HZ41 gene fragment containing the target site is obtained by PCR amplification, and sequencing is used to confirm that a tomato plant with S1HZ41 gene mutation is obtained.

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

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

[0022] Since the resistance mechanism of plants to adverse stress is very complex, improving crop resistance using traditional breeding methods often takes a long time and consumes a lot of manpower 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. Gene site editing (CRISPR / Cas) technology can accurately knock out harmful genes, and exogenous components such as vectors can be eliminated through genetic means. It is safe and efficient, and has become a research hotspot in the field 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 heat resistance and fruit size of tomatoes. The knockout vector of the SlHZ41 gene provided by the present invention can improve the POD and SOD activities of plants, inhibit the accumulation of MDA content, and reduce the damage of high temperature stress to plants; at the same time, the knockout plant increases the yield of tomatoes; and 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 It is the expression of SlHZ41 gene in different tissues of the present invention; among them, Rt: root; St: stem; YL: new leaf; OL: old leaf; LB: side branch; FL: flower; MG: green and mature; B: breaking color; B+3: breaking color for 3 days; B+7: breaking color for 7 days; B+10: breaking color for 10 days; B+15: breaking color for 15 days.

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

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

[0028] Figure 4 This is the process of constructing the pHellsgate2-SlHZ41 overexpression vector in the present invention; wherein, Figure 4 Figure A in the figure is the PCR amplification product of SlHZ41 gene; Figure 4 Figure B shows the double enzyme digestion result of pHellsgate2; Figure 4 Figure C in the figure shows the E. coli detection result of pHellsgate2-SlHZ41; Figure 4 Figure D shows the results of Agrobacterium detection of pHellsgate2-SlHZ41 recombinant plasmid.

[0029] Figure 5 is the detection result of the S1HZ41 gene knockout plant in the present invention; wherein, Figure 5 Figure A shows the SlHZ41 gene structure and target site location; Figure 5 Figure B shows the editing method of the SlHZ41 knockout site.

[0030] Figure 6 This is the detection result of the S1HZ41 gene overexpression plant in the present invention.

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

[0032] Figure 8 The knockout of S1HZ41 gene in the present invention promotes fruit ripening and fruit size; wherein, Figure 8 Figure A shows the fruit phenotypes of the second and third ears of SlHZ41 transgenic plants and wild-type plants 3 months after planting. The corresponding plant strains from left to right are WT, OE1, OE2, KO1, and KO2. Figure 8 Figure B shows the whole seedling phenotype of SlHZ41 transgenic plants and wild plants 3 months after planting. The corresponding plant strains from left to right are WT, OE1, OE2, KO1, and KO2. Figure 8 Figure C shows the fruit weight of the second and third ears of SlHZ41 transgenic plants and wild plants 3 months after planting; Figure 8 Figure D shows the pericarp thickness of the second and third ears of SlHZ41 transgenic plants and wild plants 3 months after planting; Figure 8 Figure E shows the single fruit weight of the second and third ears of SlHZ41 transgenic plants and wild plants 3 months after planting.

[0033] Fig. 9 This is the expression profile of the S1HDZ I subfamily members under heat stress in the present invention. DETAILED DESCRIPTION

[0034] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0035] Example 1

[0036] 1. Acquisition of SlHZ41 gene

[0037] Based on the previous laboratory transcriptome data analysis, the expression patterns of HD-Zip I subfamily members under heat treatment were analyzed, indicating that SlHZ41 was significantly induced by high temperature. Fig. 9 shown.

[0038] 2. High temperature treatment of tomatoes

[0039] High temperature stress: Select plump tomato 'Ailsa Craig' AC seeds, sterilize and inoculate on 20 cm × 20 cm 1 / 2MS solid medium plates. At 25 °C, relative humidity 70%, light intensity of about 800 μmol m- 2 s -1 After culturing in an artificial climate chamber for 12 days, the plates were transferred to 42°C, 70% relative humidity, and a light intensity of approximately 800 μmol m -2 s -1 The tomato seedlings were subjected to high temperature stress in a 25℃ light incubator. The tomato seedlings were sampled after being treated with high temperature for 0h, 2h, 4h, 6h, 12h, 24h, 48h, and after being treated with high temperature for 24h and then recovered for 1d in a 25℃ light incubator.

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

[0041] The formula of 1 / 2MS medium is: weigh 2.23g MS powder, dissolve in 800mL ultrapure water, add 15g sucrose, and dilute to 1000mL. Adjust pH to 5.8, add 7.5g agar, and sterilize at 121℃ for 21min. MS powder: Phytotech, M519.

[0042] In 2022, the following tissue samples were obtained from tomato plants in the fruiting period in the artificial climate chamber: Rt: root; St: stem; YL: new leaf; OL: old leaf; LB: side branch; FL: flower; MG: green and ripe fruit; B: broken color fruit; B+3: fruit after breaking color + 3 days; B+7: fruit after breaking color + 7 days; B+10: fruit after breaking color + 10 days; B+15: fruit after breaking color + 15 days; seed: seed.

[0043] Among them, "new leaves" refer to the leaves at the first and second nodes from the top of the plant to the bottom.

[0044] "Old leaves" refer to the leaves at the fourth and fifth nodes from the top of the plant.

[0045] "Green ripe" means that the tomato fruit has grown to its maximum size and will no longer expand.

[0046] "Breaking color" refers to the stage when the tomato fruit appears slightly yellow-orange and begins to mature. The total RNA of tomatoes was extracted using the NGzol total RNA rapid extraction kit from Shanghai Huiling Company according to the kit instructions and stored at -80°C for later use. Reverse transcription was performed using the HiScript II 1st Strand cDNA Synthesis Kit: +gDNAWiper from Novazon, and the quantitative kit ChamQ SYBR qPCR Master Mix was purchased from Novazon. The relative expression of genes was detected using the CFX96 fluorescent quantitative PCR instrument from Bio-Rad. Among them, the quantitative kit ChamQ SYBRqPCRMasterMix was purchased from Novazon, cat.no.Q311-01.

[0047] The expression of SlHZ41 in different tissues of AC, including roots, stems, new leaves, old leaves, lateral branches, flowers, green and ripe fruits, fruits with broken color, fruits with broken color + 3 days, fruits with broken color + 7 days, fruits with broken color + 10 days, and seeds, was determined by qRT-PCR. 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 the fruit broke color, followed by seeds, flowers, old leaves, stems and roots.

[0049] qRT-PCR was used to measure the expression of SlHZ41 at 42°C for 0h, 2h, 4h, 6h, 12h, 24h, 48h and after recovery. Figure 2 As shown, the results showed that the SlHZ41 gene was significantly induced by heat treatment at 42°C for 2h to 4h, and it was speculated that the SlHZ41 gene may be involved in the high temperature stress response process of tomato plants.

[0050] 3. Knockout vector construction

[0051] 3.1 sgRNA sequence primer design

[0052] The CRISPR-PLANT website was used to select the single-stranded guide RNA sequence of the tomato SlHZ41 gene. The sgRNA was used as the targeting guide sequence for CRISPR-Cas9 knockout, and the SlHZ41-SgRNA-F and SlHZ41-SgRNA-R oligonucleotide sequences were designed.

[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 by digesting it with BbsI overnight at room temperature, and purify and recover it after electrophoresis detection. The detection results are as follows: Figure 3 As shown in Figure A.

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

[0061] “Overnight” means time ≥ 12h.

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

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

[0064] Phosphorylated sgRNA was connected to the linearized PSgR-Cas9-At vector. Reaction system: BbsI digested psgRCas9-At 2μL, 200-fold diluted phosphorylated sgRNA 1μL, 10×T4 Ligation buffer 1μL, T4Ligase 0.25μL, ddH2O 5.75μL. After 1h of room temperature ligation reaction, E. coli was transformed by heat shock method, and the solid LB plate containing 100mg / L ampicillin antibiotic was coated with transformation. PCR detection was performed by M13F and SlHZ41-SgRNA-R. The test results are shown in Figure 3 As shown in Figure B, 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] The nucleotide sequence of M13F is shown in SEQ ID NO.8: 5'-TGTAAAACGACGGCCAGT-3'.

[0066] Heat shock method for transforming E. coli:

[0067] S1. Take out the prepared competent cells from the -80℃ refrigerator and place them on ice to slowly thaw;

[0068] S2, add 5 μL of plasmid and mix gently;

[0069] S3, ice bath for 30 min, then heat shock at 42°C for 90 s, then immediately put on ice for 2 min;

[0070] S4. Perform the operation in a clean bench and add 700 μL LB culture medium;

[0071] S5, incubate at 37℃ in a shaker for 60min;

[0072] S6. After the recovery, 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. Culture inverted in a 37°C incubator for 12 hours.

[0075] (3) Enzyme digestion of the SlHZ41-SgRNA-Cas9 PCR intermediate vector and the pCAMBIA2301 vector, wherein 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 (SlHZ41-SgRNA-Cas9 fragment as shown in Figure 3 As shown in Figure C and the pCAMBIA2301 linearized vector as Figure 3 As shown in Figure D.

[0076] The sgRNA-Cas9 fragment and the pCAMBIA2301 linearized vector were connected, wherein the reaction system: 2 μL of pCAMBIA2301 linearized vector, 4 μL of SlHZ41-SgRNA-Cas9 linearized fragment, 1 μL of 10×T4 Ligation buffer, 0.2 μL of T4 Ligase, and 2.8 μL of ddH2O. After 2 hours of connection reaction at room temperature, the competent cells of Escherichia coli were transformed by heat shock method, and colony PCR detection was performed on solid LB medium containing 50 mg / L Kana. The detection primers for colony PCR detection were pCAMBIA2301-F and SlHZ41-SgRNA-R; the nucleotide sequence of pCAMBIA2301-F is shown in SEQ ID NO.9: 5'-CGGCGAGTTCTGTTAGGTCCTCTA-3', and positive clones were screened and plasmids were extracted. The results are shown in 3E, and SlHZ41-Cas9-pCAMBIA2301 was obtained.

[0077] IV. Construction of SlHZ41 gene overexpression vector

[0078] The CDS sequence of SlHZ41:Solyc08g083130 gene was downloaded from the tomato database SGN, and the gene-specific primers OE-SlHZ41-F and OE-SlHZ41-R were designed to amplify the full-length cDNA sequence of SlHZ41 gene by PCR.

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

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

[0081] Finally, the cloned gene fragment was purified and recovered. The recovered SlHZ41 gene fragment was as follows: Figure 4 As shown in Figure A.

[0082] Construction of overexpression vector:

[0083] (1) Linearize the pHellsgate2 vector using restriction endonucleases Xho I and Xba I. Figure 4 As shown in Figure B.

[0084] (2) Connect the target gene fragment: SlHZ41 gene fragment with the linearized vector, and use the ClonExpressIIOne Step Cloning Kit homologous recombination reaction system for connection as follows: SlHZ41 gene fragment 4μL, 5×CeIIbuffer 2μL, 5×CeII Ligase 1μL, linearized pHellsgate2Vector 2μL, H2O 1μL. The reaction conditions are 37℃ in a PCR instrument for 30min. The ligation product is transformed into Escherichia coli competent cells by heat shock method. After colony PCR detection, positive clones are identified and plasmids are extracted. The results are as follows: Figure 4 As shown in Figure C, a positive plasmid was obtained and named pHellsgate2:SlHZ41.

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

[0086] The detection primers for colony PCR detection were 35S-F and OE S1HZ41-R.

[0087] The nucleotide sequence of 35S-F is shown in SEQ ID NO.10: 5'-GACGCACAATCCCACT ATCC-3'; the nucleotide sequence of OES1HZ41-R is shown in SEQ ID NO.11: 5'-TCTCATT AAAGCAGGACTCTAGACTAAAATTCCCACAATGGTGATG-3'.

[0088] The steps for heat shock transformation of E. coli competent cells are the same as above.

[0089] (3) The positive plasmid was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0090] The nucleotide sequence of the S1HZ41 gene was shown in SEQ ID NO.1:

[0091] .

[0092] The amino acid sequence of the protein expressed by the above S1HZ41 gene is shown in SEQ ID NO.2:

[0093] MAPTNSDMIWGESIMSSKCNDNMRRRFNDEQIKSLENMFETESRPELRTKQQLAKRLGLQPRQVAIWFQNKRARSKSKQLELEYRMLQISYDNLGSKYELLKKEHESLLIQLQRLKKLMEKDDNEKDVNKKSETEVKQDDFAPEFGSRGIDYLRAESDILDMAQIADGLSEIENEFNFESRTFLHDTGCTSPLWEF*.

[0094] The sequence of the target site of the above-mentioned S1HZ41 gene is shown in SEQ ID NO.3:

[0095] CAGAGTCAAGGCCTGAATTG.

[0096] 5. Agrobacterium-mediated genetic transformation of tomato

[0097] 5.1 Tomato seed treatment

[0098] Soak tomato 'Ailsa Craig' AC seeds in sterile water for 30 minutes on a clean bench. After the tomato seeds swell and absorb water, pour out the water, add 75% alcohol by volume to the triangular flask, shake for 30 seconds, pour out the alcohol, and then wash the seeds with disinfectant for 10 minutes, shaking continuously. After washing the tomato seeds with sterile ddH2O three times, inoculate the tomato seeds on 1 / 2MS culture medium and place them in an artificial climate chamber at 25℃ and 16h / 8h light for 7 days. At this time, the cotyledons of most seedlings are fully expanded.

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

[0100] 5.2 Explant preparation

[0101] Select sterile seedlings with fully expanded cotyledons on a clean bench, cut each cotyledon into two explants with a scalpel, and place the obtained tomato cotyledon nodes on a pre-culture medium, culture at 25°C in the dark for 1 day to obtain tomato explants.

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

[0103] 5.3 Agrobacterium infection and transformation of tomato

[0104] (1) GV3101 Agrobacterium tumefaciens expressing pHellsgate2:SlHZ41 or SlHZ41-Cas9-pCAMBIA2301 was streaked and activated on LB solid medium containing 50 mg / L R If and 50 mg / L Kan.

[0105] (2) Pick a single clone and add it to 15 mL of LB liquid medium containing 50 mg / L R and 50 mg / L Kan. Cultivate at 28°C with shaking until OD 600 To around 0.8.

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

[0107] (4) Place the obtained tomato explants in an empty glass dish and pour in the Agrobacterium infection solution. Shake the dish continuously for 10 minutes. Finally, transfer the tomato explants to the pre-culture medium and continue to culture in the dark for 2 days.

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

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

[0110] Agrobacterium suspension: weigh 4.43 g / L MS powder, 30 g / L sucrose, adjust pH to 5.8, and sterilize by autoclave at 121°C for 21 min. MS powder: Phytotech, M524.

[0111] 5.4 Screening and culture

[0112] Transfer the tomato explants infected with Agrobacterium and co-cultured for 2 days to the screening medium. Place about 30 explants in each culture dish with the leaves facing up. Culture them in a culture room at 25°C and 16h / 8h light for about 15 days until the callus tissue re-differentiates into adventitious buds.

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

[0114] 5.5 Subculture

[0115] The screened tomato explants were transferred back to the subculture medium and cultured in an artificial climate chamber at 25°C and 16h / 8h light intensity for about 15 days until the callus tissue redifferentiated into adventitious buds.

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

[0117] 5.6 Rooting culture

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

[0119] Among them, rooting medium: 4.43g / L MS powder, 30g / L sucrose, 7.4g / L agar, 2mg / L IBA, PH 5.8, 360mg / L TMT+50mg / L kana. Among them, MS powder: Phytotech, M519.

[0120] 5.7 Transplanting and hardening seedlings

[0121] When the adventitious roots of the tomato seedlings grow well, take out the tomato seedlings, clean the root culture medium, transplant them into a culture pot filled with vermiculite, cover them with a transparent lid to keep them moist for about a week, remove the transparent lid and harden the seedlings for 2 weeks. Finally, the transformed tomato plants can be transplanted to the greenhouse for normal field management and used for seed breeding.

[0122] Among them, normal field management is: do a good job of plant arrangement, and carry out field management such as pruning, branch cutting, vine tying, and old leaf removal in time to facilitate ventilation and light transmission.

[0123] 6. Detection of genetically modified plants

[0124] 6.1 Identification of overexpressing plants

[0125] DNA from leaves of transgenic plants and wild-type tomato plants was extracted using the CTAB method. The specific steps were as follows:

[0126] (1) Place fresh leaves in a 2 mL EP tube containing steel beads, freeze them quickly in liquid nitrogen, and grind them into powder on a grinder.

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

[0128] (3) Add 500 μL of chloroform, shake vigorously to mix, and centrifuge at 12,000 rpm for 10 min.

[0129] (4) Carefully aspirate 400 μL of the supernatant with a pipette, add an equal volume of isopropanol, shake to mix, and place in a -20°C refrigerator for precipitation reaction for about 30 min.

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

[0131] (6) Add 750 μL of 75% ethanol, shake and mix, centrifuge at 12,000 rpm for 5 min, discard the supernatant, and place 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 thoroughly, and store the DNA sample in a -20°C refrigerator.

[0133] The DNA extracted from the leaves of the transgenic plants and wild-type tomato plants was tested by PCR.

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

[0135] The PCR reaction procedure was as follows: ① pre-denaturation, 94°C for 5 min; ② denaturation, 94°C for 30 s; ③ annealing, 57°C for 30 s; ④ extension, 72°C for 1 min; ⑤ post-extension, 72°C for 5 min, wherein procedures ②, ③, and ④ were cycled 33 times.

[0136] The primers for PCR reaction were 35S-F and OE S1HZ41-R.

[0137] The nucleotide sequence of 35S-F is shown in SEQ ID NO.10; 5'-GACGCACAATCCCACT ATCC-3'; the nucleotide sequence of OES1HZ41-R is shown in SEQ ID NO.11; 5'-TCTCATTA AAGCAGGACTCTAGACTAAAATTCCCACAATGGTGATG-3'.

[0138] According to the above DNA PCR test results, 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 of Shanghai Huiling Company according to the kit instructions, the reverse transcription kit HiScript II 1st Strand cDNA Synthesis Kit: +gDNAWiper of Novazon was used, the quantitative kit ChamQ SYBR qPCR Master Mix was purchased from Novazon, and the relative expression of genes was detected using the CFX96 fluorescent quantitative PCR instrument of Bio-Rad. Among them, the quantitative kit ChamQ SYBRqPCRMasterMix was purchased from Novazon, 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 is shown in SEQ ID NO. 12; 5'-GAAGAAGGTTCAATGATGAGCAG-3'; the nucleotide sequence of SlHZ41-qPCR-R is shown in SEQ ID NO. 13; 5'-AGTCTCTGCAGCTGGATGAGG-3'.

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

[0142] qRT-PCR program: pre-denaturation: 95℃, 5min; 40 cycles: denaturation: 95℃, 10s; annealing: 60℃, 30s; extension 72℃, 30s to collect fluorescence. Tomato Actin7 was used as the internal reference gene, and each sample was repeated 3 times, 2 -ΔΔCt Calculate the relative expression.

[0143] The nucleotide sequence of Actin7-RT-F is shown in SEQ ID NO. 14: 5′-AGAAGGATGCGT ATGTGGGT-3′;

[0144] The nucleotide sequence of Actin7-RT-R is shown in SEQ ID NO.15; 5'-AGAAGGATGCGT ATGTGGGT-3'.

[0145] 6.2 Identification of CRISPR / Cas knockout plants

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

[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] 7. Heat resistance identification

[0149] Tomato AC seeds with full grains, two SlHZ41 overexpression lines with high expression levels, and SlHZ41CRISPR / Cas pure knockout lines were selected for germination in a 28℃ constant temperature incubator; white seeds were sown in nutrient pots and cultured in a 24℃ / 20℃ artificial climate box with 16h light / 8h dark. When the plants grew to four leaves and one heart, the wild-type WT, mutant plants, and overexpression plants were divided into two groups, one as the control group and the other as the experimental group. The experimental group was subjected to high temperature stress.

[0150] The nutrient pot is a mixed matrix of peat: vermiculite: perlite in a volume ratio of 3:1:1.

[0151] The method of high temperature treatment is: select tomato materials of different strains and place them randomly in an artificial climate box at 42°C, treat 15 plants of each strain, and maintain the air humidity of the climate box at about 50% during the treatment to ensure that the roots of the tomato plants have sufficient water to avoid drought stress. Record the plant phenotype, and select leaf materials of the same node of plants of different strains to measure relevant physiological and biochemical indicators; SOD activity, POD activity, malondialdehyde content and relative conductivity.

[0152] The tomato leaves before and after treatment were weighed, and then quickly frozen in liquid nitrogen. They were ground using a high-throughput sample grinder for later use.

[0153] Enzyme extraction: Add 1 mL of phosphate buffer and shake to mix thoroughly. Centrifuge at 13000g for 20 min at 4°C. The supernatant is the enzyme solution. Pipette the supernatant into a centrifuge tube and place on ice for later use.

[0154] 7.1 Determination of SOD activity

[0155] 50 mM PBS (pH = 7.8), 130 mM methionine, 750 μM nitroblue tetrazolium solution, 100 μM EDTA-Na2, 20 μM riboflavin solution and enzyme solution were added to the finger tube in sequence, and light control and dark control were set at the same time, and the absorbance of the solution was measured at a wavelength of 560 nm.

[0156] SOD activity (unit / g FW) = ((absorbance of control tube - absorbance of sample tube) * volume of extract / (absorbance of control tube * 0.5 * fresh weight of sample * amount of enzyme solution used in assay) = ((ACK-AE) * 0.8) / (ACK * 0.5 * FW * 0.1).

[0157] Wherein, ACK: absorbance of the control tube; AE: absorbance of the sample tube; 0.5: enzyme activity coefficient at half unit.

[0158] Results Figure 7 . Figure 7 (A) shows the phenotype after high temperature treatment; Figure 7 (B) in the figure is DAB staining; Figure 7 (C) in the figure shows SOD activity; Figure 7 Figure (D) shows the relative conductivity; Figure 7 Figure (E) shows the malondialdehyde content.

[0159] 7.2 Determination of POD activity

[0160] 50 mM PBS, 100 mM H2O2, 100 mM guaiacol and enzyme solution were added to a finger tube in sequence, and the absorbance of the solution was measured using a kinetic curve at a wavelength of 470 nm.

[0161] POD activity (μmol g-1s-1) = (ΔA470*0.8) / (0.015*FW*26.8*60*3) = (A470*extraction volume) / (amount of enzyme solution used in the assay*fresh weight of the sample*extinction coefficient*time).

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

[0163] The pH of PBS is 7.0.

[0164] Results Figure 7 .

[0165] 7.3 Determination of malondialdehyde content

[0166] Add enzyme solution and 0.5% thiobarbituric acid by volume into a finger-shaped tube, mix well, boil in water bath for 20 min, quickly cool on ice to room temperature, centrifuge at 3000 rpm at 4°C for 10 min, aspirate the supernatant and 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) * extract volume (mL) / plant tissue fresh weight (g).

[0169] Results Figure 7 .

[0170] 7.4 Determination of relative conductivity

[0171] Ten leaf discs were punched with a tool and placed in a glass tube containing 10 mL of deionized water. The initial conductivity (C1) was measured with a conductivity meter at 100 oscillations per minute for 2 h under normal temperature conditions. Then, the glass tube containing the leaf discs was boiled in distilled water for 30 minutes and the final conductivity (C2) was measured after the tube was cooled to ambient temperature. The conductivity (C0) of deionized water was measured and used as a blank. REC was calculated as (C1-C0) / (C2-C0)×100%.

[0172] Results Figure 7 .

[0173] Through phenotypic observation, it was found that after 24 hours of high temperature treatment, the top of the wild-type plant wilted, the overexpression plant wilted and dried up, and the knockout plant grew well. H2O2 is the main reactive oxygen species (ROS) molecule, which usually accumulates under abiotic stress. The DAB staining (detection of H2O2) method detected the accumulation of O2 and H2O2 in AC, overexpression plants and knockout plants under control and high temperature conditions. Under control conditions, there was no difference in H2O2 accumulation among all plants. Under high temperature treatment, the overexpression plants accumulated the most O2 and H2O2, the knockout plants accumulated the least H2O2, and the wild-type plants were between the two.

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

[0175] The malondialdehyde content represents the degree of membrane damage. After high temperature treatment, the malondialdehyde content in the overexpression plants was the highest, indicating that after high temperature treatment, the overexpression plants were most obviously damaged.

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

[0177] 8. Field trait statistics

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

[0179] Among them, routine management is to do a good job of plant arrangement, and timely carry out field management such as pruning, pruning, tying vines, and removing old leaves to facilitate ventilation and light transmission. Figure 8 . Figure 8 Figure A shows the fruit phenotypes of the second and third ears of SlHZ41 transgenic plants and wild-type plants 3 months after planting; Figure 8 Figure B shows the whole seedling phenotype of SlHZ41 transgenic plants and wild plants 3 months after planting; Figure 8 Figure C shows the fruit weight of the second and third ears of SlHZ41 transgenic plants and wild plants 3 months after planting; Figure 8 Figure D shows the pericarp thickness of the second and third ears of SlHZ41 transgenic plants and wild plants 3 months after planting; Figure 8 Figure E shows the single fruit weight of the second and third ears of SlHZ41 transgenic plants and wild plants 3 months after planting.

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

[0181] In summary, the statistics of fruit size and yield and overexpression of SlHZ41 reduced tomato yield, while knockout of SlHZ41 increased tomato yield.

[0182] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0183] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A S1HZ41 gene, characterized in that: The nucleotide sequence of the SlHZ41 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein expressed by the gene is shown in SEQ ID NO.

2.

2. Use of the S1HZ41 gene according to claim 1 in improving heat resistance and fruit size of tomatoes.

3. The use according to claim 2, characterized in that: By using genetic engineering means, the SlHZ41 gene is knocked out or the expression level of the SlHZ41 gene is reduced, thereby obtaining a tomato mutant with improved heat resistance and fruit size.

4. A method for improving heat resistance and fruit size of tomatoes, characterized in that: Using CRISPR / Cas9 gene editing technology to edit the target site of the SlHZ41 gene of tomato as claimed in claim 1; 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; The sequence of the target site of the SlHZ41 gene is shown in SEQ ID NO.

3.

5. The method according to claim 4, characterized in that The target site of the SlHZ41 gene is connected to a transgenic vector to construct a recombinant expression vector; the recombinant expression vector is transfected into a plant material to be transformed to obtain a gene-edited site-specific knockout tomato plant of SlHZ41.

6. The method according to claim 5, characterized in that The transgenic vector includes SlHZ41-pHellsgate2 and / or SlHZ41-Cas9-pCAMBIA2301.

7. A method for cultivating heat-resistant and fruit-size improved tomatoes, characterized in that: The following steps are involved: Obtaining the target site sequence at the nucleotide sequence of the S1HZ41 gene as claimed in claim 1, and synthesizing an sgRNA primer for the target site sequence; The sgRNA primers were used to construct the SlHZ41-Cas9-pCAMBIA2301 vector; The SlHZ41-Cas9-pCAMBIA2301 vector is transferred into Agrobacterium to obtain recombinant Agrobacterium; The recombinant Agrobacterium is transfected into tomatoes to be transformed, and tomato plants with improved heat resistance and fruit size are obtained through selection, differentiation, rooting, seedling hardening and transplanting.

8. The cultivation method according to claim 7, characterized in that: The sgRNA primer sequences include SlHZ41-SgRNA-F and SlHZ41-SgRNA-R; The nucleotide sequence of SlHZ41-SgRNA-F is shown in SEQ ID NO.4; the nucleotide sequence of SlHZ41-SgRNA-R is shown in SEQ ID NO.

5.

9. The cultivation method according to claim 7, characterized in that: After the recombinant Agrobacterium is transfected into tomatoes to be transformed, tomato S1HZ41 transgenic positive seedlings are obtained by tissue culture, S1HZ41 gene fragments containing the target site are obtained by PCR amplification, and tomato plants with S1HZ41 gene mutations are obtained by sequencing to confirm.

10. The cultivation method according to claim 9, characterized in that: The primers used in the PCR amplification include SlHZ41-check-F and SlHZ41-check-R; Among them, the nucleotide sequence of the SlHZ41-check-F is shown in SEQ ID NO.6, and the nucleotide sequence of the SlHZ41-check-R is shown in SEQ ID NO.7.

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