Application of expression inhibitor of SlBBX19 gene

Abstract

The invention belongs to the technical field of plant molecular biology, and particularly relates to application of an expression inhibitor of an SlBBX19 gene. By creating an overexpression plant and a knockout plant of the SlBBX19 gene, the SlBBX19 gene knockout plant subjected to high-temperature treatment and low-temperature treatment is found to be good in growth condition and show obvious resistance, a gene resource is provided for cultivating a new stress-resistant tomato variety, and the SlBBX19 gene knockout plant has a relatively good potential application value and has a good application prospect. And a theoretical basis is laid for researching a mechanism of responding to stress signals and a molecular mechanism of tolerating adverse environments of tomato plants.

Description

Technical Field

[0001] The invention belongs to the technical field of plant molecular biology, and particularly relates to the application of an expression inhibitor of a SlBBX19 gene. Background Art

[0002] Tomato is an important vegetable crop in the world, famous for its unique flavor and nutritional value. It is an important source of lycopene, vitamins and minerals. Tomato is a temperature-sensitive crop. Environments below 12°C and above 32°C seriously affect the yield and quality of its fruits. In facility-based tomato cultivation, plastic greenhouses account for the largest proportion because their temperature environment is poorly controllable and they are easily harmed by abiotic stresses such as low and high temperatures. Among them, the Latin name of tomato is Solanum lycopersicum L.

[0003] As one of the abiotic stresses, low temperature stress will harm the entire growth cycle of plants. Its harm is manifested as rotten seeds and low germination rate during the sowing period, leaf atrophy and growth stagnation during the seedling stage, pollen sterility and flower drop during the reproductive period, and dry matter accumulation during the fruit ripening period, resulting in reduced quality and yield. When tomatoes are exposed to high temperatures, they are very likely to grow too tall, have thin stems, thin and thin leaves, and few flower spikes and flowers. When high temperatures are encountered during the flowering and fruiting period, the floral organs are not fully developed and cannot complete normal physiological activities. The pollen vitality is low, the germination rate decreases, and it is difficult to set fruit or the fruit grows into a dead fruit. This results in low yield and poor quality of tomatoes.

[0004] In order to deal with the impact of temperature stress on tomato yield and quality, the breeding process needs to be accelerated. On the basis of traditional breeding, molecular breeding technology should be vigorously developed to explore genes with great utilization value and provide genetic resources for the cultivation of tomato germplasm that is resistant to low temperature, high temperature and other abiotic stresses.

[0005] BBX belongs to the zinc finger structural protein family and plays an important role in regulating plant growth and development and stress response. In tomatoes, SlBBX17 negatively regulates the growth of tomato plants and positively regulates heat tolerance. SlBBX31 positively regulates the cold tolerance of tomatoes by directly activating the promoters of SlCBF1 and SlCBF2. SlBBX17 cannot directly regulate SlCBFs, but physically interacts with SlHY5, thereby increasing the transcriptional activity of SlHY5 on SlCBFs genes under cold stress, positively regulating the cold tolerance of tomatoes. In apples, MdBBX37 and MdICE1 interact to form a BBX37-ICE1 complex that synergistically activates the MdCBF1 promoter and enhances cold tolerance. Given the complex functional mechanisms of BBX transcription factors in temperature stress, it is necessary to further improve their functional regulatory network. Summary of the invention

[0006] The present invention provides a new use of the SlBBX19 gene, and specifically relates to the use of an expression inhibitor of the SlBBX19 gene.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions.

[0008] The present invention provides an application of an expression inhibitor of the SlBBX19 gene in any of the following:

[0009] a. Application in improving the tolerance of tomatoes to temperature stress.

[0010] b. Application in breeding tomatoes with high tolerance to temperature stress.

[0011] The gene accession number of the SlBBX19 gene is Solyc01g110370.

[0012] The present invention studies the functional mechanism of the SlBBX19 gene in temperature stress, discovers the SlBBX19 gene and its functional regulatory relationship, and further improves the functional regulatory network of BBX transcription factors in temperature stress.

[0013] In some technical solutions of the present invention, the tolerance to temperature stress includes: tolerance to high temperature and / or tolerance to low temperature.

[0014] In some technical schemes of the present invention, the expression inhibitor of the SlBBX19 gene includes: a CRISPR / Cas9 vector for targeted knockout of the SlBBX19 gene.

[0015] In some technical schemes of the present invention, the CRISPR / Cas9 vector for targeted knockout of the SlBBX19 gene contains an sgRNA sequence targeting the SlBBX19 gene.

[0016] The sgRNA sequence includes the nucleotide sequence shown in SEQ ID NO.4 or the nucleotide sequence shown in SEQ ID NO.5.

[0017] The present invention also provides a method for improving the tolerance of tomatoes to temperature stress, comprising: inhibiting the expression of the SlBBX19 gene in tomatoes to improve the tolerance of target tomatoes to temperature stress.

[0018] The gene accession number of the SlBBX19 gene is Solyc01g110370.

[0019] In some technical schemes of the present invention, the method for inhibiting the expression of the SlBBX19 gene in tomatoes includes: using the CRISPR / Cas9 editing method to knock out the SlBBX19 gene in tomatoes.

[0020] In some technical schemes of the present invention, the vector used in the CRISPR / Cas9 editing method includes a CRISPR / Cas9 vector for targeted knockout of the SlBBX19 gene.

[0021] Targeted knockout of SlBBX19 gene The CRISPR / Cas9 vector contains a sgRNA sequence targeting the SlBBX19 gene.

[0022] The sgRNA sequence includes the nucleotide sequence shown in SEQ ID NO.4 or the nucleotide sequence shown in SEQ ID NO.5.

[0023] In some technical solutions of the present invention, the target plant includes: tomato.

[0024] In some technical solutions of the present invention, the tolerance to temperature stress includes: tolerance to high temperature and / or tolerance to low temperature.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] In the present invention, by creating overexpression plants and knockout plants of the SlBBX19 gene, it is found that the SlBBX19 gene knockout plants after high temperature treatment and low temperature treatment have good growth conditions and show obvious resistance. It is also found that after low temperature treatment, the malondialdehyde content and relative conductivity of the overexpression plants are significantly higher than those of the knockout plants; the POD activity and SOD activity of the knockout plants are significantly higher than those of the wild type and the overexpression plants, indicating that the low temperature tolerance of tomatoes is significantly improved after the SlBBX19 gene is knocked out. After high temperature treatment, the malondialdehyde content and relative conductivity of the overexpression plants are significantly higher than those of the wild type and the knockout plants; the POD activity and SOD activity of the overexpression plants are significantly lower than those of the wild type and the knockout plants, indicating that the heat resistance of tomatoes is significantly improved after the SlBBX19 gene is knocked out. The present invention provides gene resources for cultivating new stress-resistant tomato varieties, has good potential application value, and lays a theoretical foundation for studying the mechanism of tomato plants responding to stress signals and the molecular mechanism of tolerance to adverse environments. The present invention studies the functional mechanism of the SlBBX19 gene in temperature stress, discovers the SlBBX19 gene and its functional regulatory relationship, and further improves the functional regulatory network of BBX transcription factors in temperature stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the expression pattern of the SlBBX19 gene in different tissues of tomato.

[0028] Figure 2 This is the expression analysis diagram of SlBBX19 gene under low temperature and high temperature stress; Figure 2A in the figure shows the expression analysis of SlBBX19 gene under 4°C treatment; Figure 2 B in the figure shows the expression analysis of SlBBX19 gene under 42°C treatment.

[0029] Figure 3 This is a diagram of the construction process of the pHELLSgate2-SlBBX19 vector; Figure 3 A in the figure indicates the result of double enzyme digestion of pHELLSgate2 vector; Figure 3 B in the figure indicates the result of PCR amplification of SlBBX19 gene sequence; Figure 3 C in the figure represents the E. coli detection result of pHELLSgate2-SlBBX19 vector; Figure 3 D in the figure represents the results of the pHELLSgate2-SlBBX19 vector recombinant plasmid Agrobacterium detection.

[0030] Figure 4 This is a diagram of the construction process of the SlBBX19-Cas9-pBSE402 vector; Figure 4 A in the figure represents the amplification result of the target fragment; Figure 4 B in the figure indicates the result of restriction digestion of pBSE402 vector; Figure 4 C in the figure represents the results of the E. coli detection of the SlBBX19-Cas9-pBSE402 vector; Figure 4 D in the figure represents the results of Agrobacterium detection of SlBBX19-Cas9-pBSE402 vector recombinant plasmid.

[0031] Figure 5 The figure shows the acquisition and identification results of the SlBBX19 gene overexpression strain; Figure 5 A in the figure represents the pHELLSgate2-SlBBX19 vector detection result diagram; Figure 5 B in the figure represents the results of SlBBX19 gene expression detection.

[0032] Figure 6 To obtain and identify the SlBBX19 gene knockout strain; Figure 6 A in the figure indicates the construction map of SlBBX19-Cas9-pBSE402 vector and the location of sgRNA target site; Figure 6 B in the figure indicates the editing mode of the SlBBX19 gene knockout site.

[0033] Figure 7 The phenotypes of SlBBX19 gene overexpression plants and knockout plants before and after temperature stress treatment; Figure 7 A in the figure represents the plant phenotype before and after low temperature treatment; Figure 7 B in the figure shows the plant phenotype before and after high temperature treatment.

[0034] Figure 8 The figure shows the tolerance identification results of SlBBX19 gene overexpression plants and knockout plants before and after low temperature treatment; Figure 7 A in the figure represents the results of measuring the malondialdehyde content in plants before and after low temperature treatment;

[0035] Figure 7 B in the figure shows the relative conductivity measurement results of the plants before and after low temperature treatment; Figure 7 C in the figure represents the results of POD activity determination of plants before and after low temperature treatment; Figure 7 D in the figure shows the results of SOD activity measurement of plants before and after low temperature treatment.

[0036] Fig. 9 The figure shows the tolerance identification results of SlBBX19 gene overexpression plants and knockout plants before and after high temperature treatment; Figure 8 A in the figure represents the results of measuring the malondialdehyde content in plants before and after high temperature treatment;

[0037] Figure 8 B in the figure shows the relative conductivity measurement results of the plants before and after high temperature treatment; Figure 8 C in the figure represents the results of POD activity determination of plants before and after high temperature treatment; Figure 8 D in the figure represents the results of SOD activity measurement of plants before and after high temperature treatment. DETAILED DESCRIPTION

[0038] The present invention is described in detail below in conjunction with specific examples, but it 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.

[0039] Embodiment 1:

[0040] 1. Tissue specificity of SlBBX19 gene expression

[0041] Tomato tissue samples: The following tissue samples were obtained in the field in Yangling in 2022: Rt, root; St, stem; YL, young leaf; OL, old leaf; Fl, flower; MG, green maturity; Br, color breaking stage; Br+3, three days after color breaking; Br+7, seven days after color breaking; Br+10, 10 days after color breaking; Br+15, 15 days after color breaking; seed, seed.

[0042] Tomato stress samples: Tomato variety 'Ailsa Craig' was used as the test material. Seeds with full grains were selected and sterilized and inoculated on 20 cm × 20 cm 1 / 2MS solid culture medium plates. The culture medium was kept in an artificial climate chamber at 25 °C, relative humidity 70%, and light intensity of about 800 μmol m -2 s -1 After culturing for 12 days, the plates were transferred to a 4°C / 42°C light incubator under conditions of 70% relative humidity and a light intensity of approximately 800 μmol m -2 s -1 The stress treatment was carried out under the conditions of . The whole tomato seedlings treated with low temperature for 0h, 1h, 3h, 6h, 12h, 24h, Re (after 24h of low temperature treatment, recover in a 25℃ light incubator for 1d), and high temperature treatment for 0h, 2h, 4h, 6h, 12h, 24h, Re (after 24h of high temperature treatment, recover in a 25℃ light incubator for 1d) were sampled. The formula of 1 / 2MS culture medium is: weigh 2.23g MS powder, dissolve in 800mL ultrapure water, add 15g sucrose, and dilute to 1000mL. Adjust the pH to 5.8, agar 7.5g, and autoclave at 121℃ for 21min.

[0043] Among them, 'Ailsa Craig' is referred to as AC. This material was bred by Professor Zhan Xiangqiang's team from the Laboratory of Stress Biology of Horticultural Crops at Northwest Agriculture and Forestry University.

[0044] MS powder: Phytotech, M519.

[0045] TRIzol (Tiangen) was used to extract RNA from all samples, and the operation method was carried out according to the instructions of the kit. The first-strand cDNA was synthesized using HiScript II 1st Strand cDNA Synthesis Kit, and the operation method was carried out according to the instructions of the kit. Quantitative primers were designed using Primer Premier 5.0 software, and gene expression was detected using Power SYBR Green PCR MasterMix. Real-time quantitative RT-PCR was performed on BIO-RAD CFX384Touch.

[0046] qPCR reaction conditions: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 10 s, 40 cycles; 72°C for 5 min.

[0047] qPCR reaction system, 15 μL: ddH2O 4.9 μL, SlBBX19-RT-F 0.3 μL, SlBBX19-RT-R 0.3 μL, Taq SYBR Green qPCRPremix (Universal) 7.5 μL, cDNA 2 μL.

[0048] Primer sequences:

[0049] The nucleotide sequence of SlBBX19-RT-F is shown in SEQ ID NO.19:

[0050] 5′-GGCAGAGGATTGAGTTTCCG-3′.

[0051] The nucleotide sequence of SlBBX19-RT-R is shown in SEQ ID NO.20:

[0052] 5'-CTCCCTGTTTTGGTTGGTGATT-3'.

[0053] 2. Construction of overexpression vector

[0054] 1. SlBBX19 gene cloning

[0055] The CDS sequence of the SlBBX19 gene was downloaded from the tomato genome database SGN, and specific primers OE-SlBBX19-F and OE-SlBBX19-R were designed. The primer sequences are shown in SEQ ID NO.1 to SEQ ID NO.2 in Table 1. The full-length cDNA sequence of SlBBX19 was amplified, and the cloned gene fragment was purified and recovered (as shown in Table 1). Figure 3 (as shown in B in the figure).

[0056] The website of the database SGN is https: / / solgenomics.net / .

[0057] The gene accession number of the SlBBX19 gene is Solyc01g110370.

[0058] The size of the CDS sequence of the SlBBX19 gene is 726 bp.

[0059] PCR reaction conditions: 95°C for 5 min; 95°C for 30 s, 57°C for 30 s, 72°C for 1 min, 35 cycles; 72°C for 5 min.

[0060] PCR reaction system, 25μL: ddH2O 7.5μL, 2×Phanta Max Master Buffer 12.5μL, OE-SlBBX19-F 1μL, OE-SlBBX19-R 1μL, dNTP 0.5μL, template cDNA 2μL, Phanta Max Super-Fidelity DNA Polymerase 0.5μL.

[0061] 2. Construction of overexpression vector:

[0062] (1) Linearize the pHELLSgate2 vector using restriction endonucleases Xho I and Xba I. Figure 3 As shown in A.

[0063] (2) Use homologous recombination to connect the gene fragment and the linearized vector.

[0064] Reaction system: SlBBX19 gene target fragment, 3μL, linearized pHELLSgate2 Vector 2μL, LightNing DNAAssembly Mix Plus 5μL.

[0065] Reaction conditions: 50°C for 60 min.

[0066] The ligation product was transformed into E. coli by heat shock method. Colony PCR was performed using 35S-F and OE-SlBBX19-R primers. The positive clone plasmid was extracted and sent to the company for sequencing using 35S-F primer. The positive clone plasmid with correct sequencing was named pHELLSgate2-SlBBX19. The test results are as follows Figure 3 As shown in C.

[0067] Table 1 SlBBX19 gene cloning primers and detection primers

[0068]

[0069] 3. Knockout vector construction

[0070] 1. sgRNA sequence primer design

[0071] According to the genome sequence of SlBBX19, CRISPR RGEN Tools was used to design the sgRNA dual-target sequence, and the sgRNA1 sequence and sgRNA2 sequence were selected as the targeting guide sequence for CRISPR-Cas9 knockout, and the primer sequence was designed according to the targeting guide sequence.

[0072] Among them, the website of CRISPR RGEN Tools is http: / / www.rgenome.net / .

[0073] The sgRNA1 sequence and the sgRNA2 sequence are shown in SEQ ID NO.4 to SEQ ID NO.5 in Table 2, and the primer sequences are shown in SEQ ID NO.6 to SEQ ID NO.7 in Table 2.

[0074] Table 2 sgRNA target sequences and sgRNA target primer sequences

[0075]

[0076] 2. Construction of CRISPR-Cas9 knockout vector

[0077] (1) Enzyme digestion of pBSE402 vector: 0.5 μL Bsa I, 10 μL pBSE402 vector, 2 μL Cut SmartBuffer, 7.5 μL ddH2O. Digestion at 37°C for 1 h. After electrophoresis, the linearized vector was recovered using the Tiangen DNA purification kit. The test results are as follows: Figure 4 As shown in B.

[0078] (2) PCR product amplification: The intermediate vector pCBC-DT1T2 was used as a template and SlBBX19-sgRNA-F and SlBBX19-sgRNA-R were used for PCR amplification. The amplified fragment included sgRNA1 / U6-26t / U6-29p / sgRNA2, and the fragment size was 622 bp.

[0079] Reaction system: ddH2O 7.5μL, 2×Phanta Max Master Buffer 12.5μL, SlBBX19-sgRNA-F 1μL, SlBBX19-sgRNA-R 1μL, dNTP 0.5μL, template pCBC-DT1T22μL, Phanta Max Super-Fidelity DNA Polymerase 0.5μL.

[0080] The reaction procedure was: 95℃5min; 95℃30s, 57℃30s, 72℃1min, 35 cycles; 72℃5min. The PCR product was detected by electrophoresis and recovered by gel cutting. The results were as follows: Figure 4 As shown in A.

[0081] (3) sgRNA was connected to the linearized pBSE402 vector. The reaction system was: 3 μL of sgRNA target fragment, 2 μL of linearized pBSE402 Vector, and 5 μL of LightNing DNAAssembly Mix Plus. After the connection reaction at 50°C for 1 hour, Escherichia coli was transformed by the heat shock method. The screening antibiotic was 100 mg / L kanamycin. The PCR positive detection was performed using primers U626-F and U629-R, and the band size was 626 bp. The recombinant plasmid of the positive clone was extracted and sent to the company for sequencing using EDIT-3 primers. The correctly sequenced recombinant plasmid was named SlBBX19-Cas9-pBSE402. The sequences of primers U626-F, U629-R and EDIT-3 are shown in SEQ ID NO.8 to SEQ ID NO.10 in Table 3. The test results are shown in Figure 4 As shown in C.

[0082] Table 3 Knockout vector detection primers and sequencing primers

[0083]

[0084] IV. Obtaining SlBBX19 gene overexpression plants and knockout plants:

[0085] 4.1 Tomato seed treatment

[0086] Soak tomato 'Ailsa Craig' AC seeds in sterile water for 30 minutes in 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 84 disinfectant and sterile water in a volume ratio of 1:1 for 8 minutes, shaking continuously during the period. Pour out the disinfectant, wash the tomato seeds twice with sterile ddH2O, and then inoculate the tomato seeds on 1 / 2MS culture medium, and place them in an artificial climate chamber at 25℃ / 22℃ and 16h / 8h light for 7 days, at which time the cotyledons of most seedlings are fully expanded.

[0087] 4.2 Explant preparation

[0088] Select sterile seedlings with fully expanded cotyledons in an ultra-clean workbench, 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.

[0089] 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, add 7.5 g / L agar, and sterilize at 121°C for 21 minutes.

[0090] Among them, MS powder: Phytotech, M524.

[0091] 4.3 Agrobacterium infection and transformation of tomato

[0092] (1) Take GV3101 Agrobacterium containing pHELLSgate2-SlBBX19 or SlBBX19-Cas9-pBSE402 and streak it on LB solid medium containing 50 mg / L Rif and 50 mg / L Kan for activation.

[0093] (2) Pick a single clone and add it to 15 mL of LB liquid medium containing 50 mg / L Rif and 50 mg / L Kan. Cultivate at 28°C with shaking until OD 600 =about 0.8.

[0094] (3) Collect the bacteria by centrifugation, add an equal volume of Agrobacterium suspension, and add 100 mmol / L AS to prepare Agrobacterium infection solution.

[0095] (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.

[0096] Among them, LB liquid culture medium: yeast extract 5g / L, tryptone 5g / L, sodium chloride 10g / L; LB solid culture medium: yeast extract 5g / L, tryptone 5g / L, sodium chloride 10g / L and agar 15g / L.

[0097] Agrobacterium suspension: weigh 4.43 g / L MS powder and 30 g / L sucrose, adjust the pH to 5.8, and sterilize at 121°C for 21 min.

[0098] Among them, MS powder: Phytotech, M524.

[0099] 4.4 Screening and culture

[0100] 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.

[0101] Among them, the screening culture medium: 4.43g / L MS powder, 30g / L sucrose, 7.4g / L agar, 0.1mg / L IAA, 2mg / L zeatin ZR, 100mg / L kanamycin Kana and 360mg / L timentin, pH 5.8.

[0102] Among them, MS powder: Phytotech, M519.

[0103] Kanamycin Kana was used to screen explants carrying the SlBBX19-Cas9-pBSE402 vector, and 10% by weight of Basta antibiotic was used.

[0104] 4.5 Subculture

[0105] 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.

[0106] Among them, the subculture culture 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.

[0107] Among them, MS powder: Phytotech, M519.

[0108] Kanamycin Kana was used for subculture of explants of SlBBX19-Cas9-pBSE402 vectors, while 10% by mass of Basta antibiotic was used.

[0109] 4.6 Rooting culture

[0110] When the adventitious buds grew to 2 cm, they were cut off from the base and transferred to rooting medium to induce rooting.

[0111] Among them, rooting medium: 4.43g / LMS powder, 30g / L sucrose, 7.4g / L agar, 2mg / L IBA, PH 5.8, 360mg / L TMT+50mg / L kana.

[0112] Among them, MS powder: Phytotech, M519.

[0113] 4.7 Transplanting and hardening seedlings

[0114] 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.

[0115] 5. Identification of transgenic plants

[0116] 1. Identification of gain-of-function mutant plants

[0117] The DNA of the leaves of the tomato plants to be tested and the wild-type tomato plants was extracted by the CTAB method. The specific operation was as follows:

[0118] (1) Place fresh leaves in a 2 mL centrifuge tube filled with steel beads, freeze them in liquid nitrogen, and grind them into powder.

[0119] (2) Add 600 μL of C TAAB extract, vortex to mix, and place in a 65°C water bath for 30 min.

[0120] (3) Add 600 μL of chloroform in a fume hood, shake to mix, and centrifuge at 12,000 rpm for 10 min.

[0121] (4) Carefully aspirate 400 μL of the supernatant using a 1 mL pipette and transfer it to a new centrifuge tube. Add an equal volume of isopropanol, mix well, and place in a -20 °C refrigerator to allow sedimentation reaction for about 30 min.

[0122] (5) Centrifuge at 12000 rpm for 10 min and discard the supernatant.

[0123] (6) Add 1 mL of 75% by volume ethanol, shake and mix, centrifuge at 12,000 rpm for 5 min, discard the supernatant, and place in a fume hood to evaporate the ethanol.

[0124] (7) Finally, add 100 μL Tris-HCl solution, mix thoroughly, and store the DNA sample in a -20°C refrigerator. Tris-HCl: 10 Mm, pH 8.0.

[0125] Identification of gain-of-function mutants by PCR:

[0126] Reaction system: 5 μL 2×Taq Master Mix, 0.2 μL 10 μM 35S-F, 0.2 μL 10 μM OE-SlBBX19-R, 2 μL DNA, 2.6 μL ddH2O.

[0127] The PCR program was: 95°C for 5 min; 95°C for 30 s, 57°C for 30 s, 72°C for 1 min, 32 cycles; 72°C for 5 min.

[0128] According to the DNA test results, DNA-positive plants were selected for RNA level detection. The specific operations were as follows: TRIzol (Tiangen) was used to extract RNA from all samples, and the operation method was carried out according to the instructions of the kit. HiScript II 1st StrandcDNA Synthesis Kit (Vazyme) was used to synthesize the first-strand cDNA, and the operation method was carried out according to the instructions of the kit. Quantitative primers were designed using Primer Premier 5.0 software, and Power SYBR Green PCR Master Mix (Applied Biosystems) was used to detect gene expression. Real-time quantitative RT-PCR (qPCR) was completed on BIO-RAD CFX384 Touch. The quantitative primer sequences are shown in SEQ ID NO.11 to SEQ ID NO.14 in Table 4.

[0129] The RT-qPCR reaction system used: 2×ChamQ Universal SYBR qPCR Master Mix 7.5μL, 0.3μL 10μM SlBBX19-qPCR-F, 0.3μL 10μM SlBBX19-qPCR-R, 2μL cDNA, and 4.9μL ddH2O.

[0130] The RT-qPCR program was: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 10 s (collecting fluorescence), 40 cycles; 72°C for 5 min. The tomato SlACT7 gene was used as the internal reference gene, and each sample was repeated three times. -ΔΔCt The data were calculated using the fluorescence threshold method and analyzed using Microsoft Excel 2019.

[0131] Among them, the accession number of SlACT7 gene is: Solyc11g005330.

[0132] Table 4 Quantitative primers

[0133]

[0134]

[0135] 2. Identification of loss-of-function mutant plants

[0136] DNA from leaves of transgenic plants and wild-type tomato plants was extracted by CTAB method, and positive seedlings were detected using EDIT-3 and SlBBX19-SgRNA-R primers. Sequencing primers were designed around 300 bp before and after the target sites sgRNA1 and sgRNA2 of the SlBBX19 gene using PrimerPremier 5.0 software: SlBBX19-check1-F, SlBBX19-check1-R and SlBBX19-check2-F, SlBBX19-check2-R. DNA from plants with positive DNA detection was used as a template to amplify fragments before and after the sgRNA target site. The PCR products were sent to the company for sequencing and compared with the reference sequence to determine whether a mutation occurred. The sequencing primer sequences are shown in SEQ ID NO.15 to SEQ ID NO.18 in Table 5.

[0137] Table 5 Sequencing primers

[0138]

[0139] VI. Temperature stress tolerance identification

[0140] The seeds of wild-type AC with full grains, two overexpression strains with the highest expression level, and two CRISPR / Cas9 knockout strains were selected, germinated at 25℃, and sown in 6.5cm diameter seedling cups. The culture conditions were: alternating light and dark culture, daytime temperature of 25℃, light intensity of 20000lux, culture for 16h, night temperature of 20℃, culture for 8h. When the seedlings grew to five leaves and one heart, they were used for subsequent experimental treatments.

[0141] 1. Low temperature treatment: When the tomato seedlings grow to five leaves and one heart, select healthy plants of similar size, treat 15 of each strain, and randomly divide them into two groups. One group is placed in an artificial climate box for constant temperature culture at 4℃, and the other group is placed in 25℃ for control. Treat at 4℃ for 7 days, with a photoperiod of 16h / 8h, a light intensity of 20,000lux, and an environmental relative humidity of 70%. Observe the plant phenotype, and measure the relevant physiological and biochemical indicators of plants of different strains, including relative conductivity, malondialdehyde content, POD and SOD activity.

[0142] 2. High temperature treatment: Select healthy plants of different strains with similar sizes and place them in an artificial climate box for 48 hours at 42℃ as the experimental group, and the other group is placed at 25℃ as the control group. The experimental group and the control group are cultured in alternating light and dark at the same time, with a photoperiod of 16h / 8h, a light intensity of 20,000lux, and a relative humidity of about 50%. Observe the plant phenotype, and measure the relevant physiological and biochemical indicators of plants of different strains.

[0143] 3. Determination of relative conductivity: Add 10 ml of ddH2O to a 15 mL centrifuge tube, and then add 10 leaves with a diameter of 1 cm to the centrifuge tube. Place the centrifuge tube in a 28 ° C shaker for 2 hours and measure the initial conductivity (EC1). Subsequently, boil the sample in water for 30 minutes, cool to room temperature, and measure the final conductivity (EC2). The calculation formula for relative conductivity (REL) is REL = EC1 / EC2 × 100%.

[0144] 4. Determination of malondialdehyde content and enzyme activity:

[0145] (1) Enzyme extraction:

[0146] 1) Weigh 0.1g of tomato leaves using a 1 / 10,000 balance and place in a pre-cooled mortar. Remove the veins of the tomato leaves as much as possible.

[0147] 2) Transfer the ground homogenate to a centrifuge tube and dilute to 1 mL with phosphate buffer; then place the centrifuge tube in an ice box and wait for centrifugation.

[0148] 3) Centrifuge at 12000 rpm at 4°C for 20 min. The supernatant is the enzyme solution.

[0149] 4) After centrifugation, keep the sample in an ice box and immediately measure the activity of antioxidant enzymes.

[0150] (2) Determination of malondialdehyde content

[0151] 0.5% TBA by volume: Weigh 5 g of trichloroacetic acid and dissolve it in 100 mL of water. Then weigh 0.5 g of TBA and dissolve it in 10 mL of 1M NaOH, and finally dilute to 100 mL with the freshly prepared 5% trichloroacetic acid solution.

[0152] The NaOH solution was prepared by dissolving 4 g in 100 mL of water.

[0153] This reagent should be prepared and used immediately, placed at room temperature, and protected from light. Add 150 μL of 0.5% TBA and 75 μL of enzyme solution to a 1.5 mL centrifuge tube, mix well, and boil in a boiling water bath for 20 minutes. When the time is up, immediately cool to room temperature in an ice water bath, centrifuge at 3000 rpm for 10 minutes, take the supernatant, and measure the absorbance at 532 nm, 600 nm, and 450 nm using a UV spectrophotometer.

[0154] The calculation formula is:

[0155] MDA concentration (μmol / L) = 6.45 (OD 532nm -OD 600nm )-0.56×OD 450nm .

[0156] MDA content (μmol / g fresh weight) = MDA concentration (μmol / L) × extract volume (mL) / plant tissue fresh weight (g).

[0157] 5. Determination of POD activity

[0158] 50mM (pH 7.0) phosphate buffer; 100mM H2O2: 1021μL 30% H2O2, dilute to 100mL, store at 4℃ away from light, use immediately after preparation; 100mM guaiacol: draw 0.4mL dilute to 100mL, store at 4℃. Add 230μL 50mM (pH 7.0) phosphate buffer, 30μL 100mM guaiacol, 15μL enzyme solution, and 30μL 100mM H2O2 in sequence to start the reaction. The measurement wavelength is 470nm, and the kinetic curve is used.

[0159] The calculation formula is:

[0160] POD activity (μmol g -1 s -1 )=(A 470nm × 0.8) / (0.015×FW×26.8×60×3)=(absorbance value×volume of extract) / (amount of enzyme solution used in the assay×fresh weight of sample×extinction coefficient×time).

[0161] 6. Determination of SOD activity

[0162] When measuring SOD, place the experimental reaction solution in a finger-shaped tube, add 160μL of 0.05mol / L phosphate buffer (pH7.8), 30μL of 130mmol / L methionine (Met) solution, 30μL of 750μmol / L nitroblue tetrazolium solution, 30μL of 100μmol / L EDTA-Na2 solution, 20μL of enzyme solution and 30μL of 20μmol / L riboflavin solution to the sample tube in sequence. Add 180μL of 0.05mol / L phosphate buffer (pH7.8) to the control tube without enzyme solution, and the rest of the reagents and order are the same as those in the sample tube. Measure the absorbance value at a wavelength of 560nm.

[0163] The calculation formula of SOD activity is:

[0164] SOD activity = ((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 the assay).

[0165] 7. Experimental results

[0166] (1) The SlBBX19 gene is expressed in all tissues of tomato.

[0167] like Figure 1 As shown, qRT-PCR detection revealed that the SlBBX19 gene was expressed in various tissues and organs of tomato AC (roots, stems, leaves, flowers, fruits, seeds, etc.).

[0168] (2) The SlBBX19 gene was significantly induced by low and high temperatures.

[0169] The expression level of SlBBX19 gene was detected by qRT-PCR at 4℃ (0h, 1h, 3h, 6h, 12h, 24h and after recovery) and 42℃ (0h, 2h, 4h, 6h, 12h, 24h and after recovery). Figure 2 A and B in Figure 1), the SlBBX19 gene showed a significant upward trend under low temperature stress and reached a maximum value at 24h; the SlBBX19 gene showed a significant downward trend under high temperature stress and reached a minimum value at 2h. Therefore, it is speculated that the tomato SlBBX19 gene is involved in the response to low and high temperature stress.

[0170] (3) Figure 3 As shown, pHELLSgate2-SlBBX19 and SlBBX19-Cas9-pBSE402 vectors were successfully constructed.

[0171] (4) Successfully created knockout and overexpression mutant plants

[0172] DNA was extracted and transgenic tests were performed, and 11 transgenic positive lines were obtained (see Figure 5 A and B in Figure 1). RNA was extracted from leaves of positive lines and tested by qRT-PCR. Fluorescence quantitative results showed that the expression level of SlBBX19 increased to varying degrees in multiple transgenic lines. OE-7 and OE-12 were selected for subsequent experiments and seed propagation.

[0173] Two knockout lines were obtained by sequencing analysis (see Figure 6 A and B in the figure), it was found that the KO5 line had an 8bp base deletion at the second target site. The KO5 line was a homozygous mutation, which caused the target gene to shift in frame, the stop codon to appear early, and the protein translation to terminate early. The KO41 line had two editing methods at the second target site, one allele had a 1bp base insertion, and the other allele had an 8bp base deletion. Both target genes had a frame shift, the stop codon appeared early, and the protein translation terminated early. Both knockout strains can be used for subsequent experiments and breeding.

[0174] (5) Knocking out the SlBBX19 gene significantly improves the low temperature tolerance of tomato plants

[0175] The phenotypic observations revealed (see Figure 7A), after 7 days of low temperature treatment, the overexpression plants wilted severely, and the whole plant turned yellow and dried up, while the knockout plants grew well and showed obvious resistance. By measuring the malondialdehyde content and relative conductivity, the degree of damage to plants under abiotic stress can be understood. Therefore, after low temperature treatment, the malondialdehyde content and relative conductivity of the overexpression plants were significantly higher than those of the knockout plants, indicating that the degree of damage to the overexpression plants was more serious. Enzyme activity assays found that after low temperature treatment, the POD activity and SOD activity of the knockout plants were significantly higher than those of the wild type and overexpression plants, indicating that the knockout strain has a stronger ability to scavenge reactive oxygen species, causing the knockout plants to suffer less reactive oxygen damage (see Figure 7 In summary, knocking out the SlBBX19 gene significantly improved the low temperature tolerance of tomato.

[0176] (6) Knocking out the SlBBX19 gene improves the heat tolerance of tomatoes

[0177] The phenotypic observations revealed (see Figure 8 A), after high temperature treatment for 48h, the overexpression plants wilted severely, dried up and turned yellow, while the knockout plants grew well. Moreover, after high temperature treatment, the malondialdehyde content and relative conductivity of the overexpression plants were significantly higher than those of the wild type and knockout plants; the POD activity and SOD activity of the overexpression plants were significantly lower than those of the wild type and knockout plants (see Figure 8 The above indicates that knocking out the SlBBX19 gene significantly improved the heat tolerance of tomatoes.

[0178] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0179] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of an expression inhibitor of the SlBBX19 gene in any of the following, characterized in that: a. Application in improving the tolerance of tomatoes to temperature stress; b. Application in breeding tomatoes with high tolerance to temperature stress; The gene accession number of the SlBBX19 gene is Solyc01g110370.

2. The use according to claim 1, characterized in that The tolerance to temperature stress includes: tolerance to high temperature and / or tolerance to low temperature.

3. The use according to claim 1, characterized in that The expression inhibitor of the SlBBX19 gene includes: a CRISPR / Cas9 vector for targeted knockout of the SlBBX19 gene.

4. The use according to claim 3, characterized in that The CRISPR / Cas9 vector for targeted knockout of the SlBBX19 gene contains an sgRNA sequence targeting the SlBBX19 gene; The sgRNA sequence includes the nucleotide sequence shown in SEQ ID NO.4 and / or the nucleotide sequence shown in SEQ ID NO.

5.

5. A method for improving the tolerance of tomatoes to temperature stress, characterized in that: include: Improving the tolerance of tomatoes to temperature stress by inhibiting the expression of the SlBBX19 gene as claimed in claim 1 in tomatoes; The gene accession number of the SlBBX19 gene is Solyc01g110370.

6. The method according to claim 5, characterized in that The method for inhibiting the expression of the SlBBX19 gene in tomatoes includes: using the CRISPR / Cas9 editing method to knock out the SlBBX19 gene in tomatoes.

7. The method according to claim 6, characterized in that The vector used in the CRISPR / Cas9 editing method includes a CRISPR / Cas9 vector for targeted knockout of the SlBBX19 gene; The CRISPR / Cas9 vector for targeted knockout of the SlBBX19 gene contains an sgRNA sequence targeting the SlBBX19 gene; The sgRNA sequence includes the nucleotide sequence shown in SEQ ID NO.4 or the nucleotide sequence shown in SEQ ID NO.

5.

8. The method according to claim 5, characterized in that The tolerance to temperature stress includes: tolerance to high temperature and / or tolerance to low temperature.

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