Use of an expression inhibitor of slbbx19 gene

Abstract

The application belongs to the technical field of plant molecular biology, and particularly relates to application of an expression inhibitor of SlBBX19 gene. In the application, overexpression plants and knockout plants of the SlBBX19 gene are created, and it is found that the growth condition of the SlBBX19 gene knockout plants after high-temperature treatment and low-temperature treatment is good, and the plants show obvious resistance, so that gene resources are provided for cultivating new tomato varieties with resistance, and have good potential application value, and lay a theoretical foundation for researching a mechanism of tomato plants responding to adversity signals and a molecular mechanism of tolerating adverse environments.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology technology, specifically involving SlBBX19 The application of gene expression inhibitors. Background Technology

[0002] Tomatoes are a globally important vegetable crop, renowned for their unique flavor and nutritional value, and are a significant source of lycopene, vitamins, and minerals. Tomatoes are temperature-sensitive; temperatures below 12°C and above 32°C severely impact fruit yield and quality. In protected tomato cultivation, plastic greenhouses account for the largest proportion due to their poor temperature control, making them susceptible to damage from abiotic stresses such as low and high temperatures. The Latin name for tomato is... Solanum lycopersicum L.

[0003] Low temperature stress, as an abiotic stress, can harm plants throughout their entire growth cycle. Its effects manifest as seed rot and low germination rates during the sowing period; leaf withering and growth stagnation during the seedling stage; pollen sterility and flower drop during the reproductive stage; and reduced dry matter accumulation, leading to decreased quality and yield during fruit ripening. Tomatoes, in particular, are prone to excessive vegetative growth, resulting in weak stems, thin leaves, and reduced flower spikes and number of flowers when exposed to high temperatures. High temperatures during the flowering and fruiting period cause incomplete flower development, hindering normal physiological activities, resulting in low pollen viability, decreased germination rates, and poor fruit set or the formation of stunted fruits. This ultimately leads to low tomato yield and poor quality.

[0004] To address the bottleneck issue in tomato germplasm and mitigate the impact of temperature stress on tomato yield and quality, it is necessary to accelerate the breeding process. Building upon traditional breeding methods, we must vigorously develop molecular breeding techniques to identify genes with significant utilization value, providing genetic resources for cultivating tomato germplasm resistant to abiotic stresses such as low temperatures and high temperatures.

[0005] BBX belongs to the zinc finger structural protein family and plays an important role in regulating plant growth, development, and stress responses. In tomatoes, SlBBX17 It negatively regulates the growth of tomato plants and positively regulates heat resistance. SlBBX31 Cold resistance of tomatoes can be positively regulated by directly activating the promoters of SlCBF1 and SlCBF2. SlBBX17 SlCBFs cannot be directly regulated; instead, they interact physically with SlHY5, thereby enhancing... SlHY5 Under cold stress, the transcriptional activity of SlCBFs genes positively regulates the cold tolerance of tomatoes. In apples, MdBBX37 and MdICE1 interact to form the BBX37-ICE1 complex, which synergistically activates the MdCBF1 promoter, enhancing cold tolerance. Given the complex functional mechanisms of BBX transcription factors under temperature stress, further research is needed to refine their functional regulatory network. Summary of the Invention

[0006] This invention provides SlBBX19 New uses for genes, specifically involving SlBBX19 The application of gene expression inhibitors.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution.

[0008] This invention provides SlBBX19 The application of gene expression inhibitors in any of the following:

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

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

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

[0012] This invention, through the SlBBX19 The study investigated the functional mechanisms of genes under temperature stress and discovered... SlBBX19 The study investigated the relationship between genes and their functional regulation, thereby further refining the functional regulatory network of BBX transcription factors under 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 solutions of the present invention, the... SlBBX19 Gene expression inhibitors include: targeted knockout SlBBX19 CRISPR / Cas9 vector for genes.

[0015] In some technical solutions of this invention, targeted knockout SlBBX19 The CRISPR / Cas9 vector of the gene contains a target SlBBX19 The sgRNA sequence of the gene.

[0016] The sgRNA sequence includes a nucleotide sequence as shown in SEQ ID NO. 4 or a nucleotide sequence as 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 growth of certain substances in tomatoes... SlBBX19 Gene expression was modified to improve the tolerance of target tomatoes to temperature stress.

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

[0019] In some technical solutions of this invention, the inhibition of the aforementioned in tomatoes SlBBX19 Methods for gene expression include: using CRISPR / Cas9 editing to specifically knock out the gene described in tomatoes. SlBBX19 Gene.

[0020] In some technical solutions of this invention, the vector used in the CRISPR / Cas9 editing method includes targeted knockout. SlBBX19 CRISPR / Cas9 vector for genes.

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

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

[0023] In some technical solutions of this 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 this invention, by creating SlBBX19 In overexpressing and knockout plants of the gene, it was found that high-temperature and low-temperature treatments resulted in... SlBBX19 The gene knockout plants grew well and showed significant resistance. Furthermore, after low-temperature treatment, the malondialdehyde (MDA) content and relative conductivity of the overexpressing plants were significantly higher than those of the knockout plants; the POD and SOD activities of the knockout plants were significantly higher than those of the wild-type and overexpressing plants, indicating that the knockout plants... SlBBX19 Genetic modification significantly improved the low-temperature tolerance of tomatoes. After high-temperature treatment, the malondialdehyde (MDA) content and relative conductivity of overexpressing plants were significantly higher than those of wild-type and knockout plants; the POD and SOD activities of overexpressing plants were significantly lower than those of wild-type and knockout plants, indicating that the knockout gene significantly increased the tolerance of tomatoes. SlBBX19 Genetic modification significantly improved the heat tolerance of tomatoes. This invention provides genetic resources for breeding new stress-resistant tomato varieties, has good potential application value, and lays a theoretical foundation for studying the mechanisms by which tomato plants respond to stress signals and the molecular mechanisms of tolerance to adverse environments. This invention, through the modification of genetic modification genes, significantly improves the heat tolerance of tomatoes. SlBBX19 The study investigated the functional mechanisms of genes under temperature stress and discovered... SlBBX19 The study investigated the relationship between genes and their functional regulation, thereby further refining the functional regulatory network of BBX transcription factors under temperature stress. Attached Figure Description

[0027] Figure 1 for SlBBX19 A diagram showing gene expression patterns in different tissues of a tomato.

[0028] Figure 2 for SlBBX19 Gene expression analysis diagrams under low temperature and high temperature stress; among them, Figure 2 In this context, A represents the temperature at 4℃. SlBBX19 Gene expression analysis diagram; Figure 2 In this context, B indicates the temperature at 42℃. SlBBX19 Gene expression analysis diagram.

[0029] Figure 3 For pHELLSgate2- SlBBX19 Carrier construction process diagram; among which, Figure 3 In the image, A represents the result of double digestion of the pHELLSgate2 vector; Figure 3 B in the text represents SlBBX19 Image of gene sequence PCR amplification results; Figure 3 The C in the text represents pHELLSgate2- SlBBX19 Image showing the results of E. coli detection in vectors; Figure 3 In this context, D represents pHELLSgate2- SlBBX19 Image showing the detection results of Agrobacterium-mediated recombinant plasmid vector.

[0030] Figure 4 This is a diagram illustrating the construction process of the SlBBX19-Cas9-pBSE402 vector; where, Figure 4 In the image, A represents the amplification result of the target fragment; Figure 4 In the image, B represents the enzyme digestion result of the pBSE402 vector; Figure 4 In the image, C represents the detection result of E. coli using the SlBBX19-Cas9-pBSE402 vector; Figure 4 The "D" in the figure represents the Agrobacterium detection result of the SlBBX19-Cas9-pBSE402 vector recombinant plasmid.

[0031] Figure 5 for SlBBX19 Figure showing the results of obtaining and identifying gene overexpression lines; among them, Figure 5 In the figure, A represents the detection results of the pHELLSgate2-SlBBX19 vector; Figure 5 B in the text represents SlBBX19 Image showing the results of gene expression level detection.

[0032] Figure 6 for SlBBX19 Obtaining and identifying gene knockout lines; among which, Figure 6In this diagram, A represents the construction map of the SlBBX19-Cas9-pBSE402 vector and the location of the sgRNA target site; Figure 6 B in the text represents SlBBX19 Editing methods for gene knockout sites.

[0033] Figure 7 for SlBBX19 Phenotypic images of overexpressing and knockout plants before and after temperature stress treatment; among them, Figure 7 In the diagram, A represents the plant phenotypic diagram before and after low-temperature treatment; Figure 7 In the diagram, B represents the plant phenotypic diagram before and after high-temperature treatment.

[0034] Figure 8 for SlBBX19 The results of the tolerance assessment of gene-overexpressing and knockout plants before and after low-temperature treatment are shown in the figure; among them, Figure 8 In the figure, A represents the results of malondialdehyde (MDA) content determination in plants before and after low-temperature treatment. Figure 8 In the figure, B represents the results of the relative electrical conductivity measurement of the plants before and after low temperature treatment; Figure 8 The C in the figure represents the results of POD activity measurement of plants before and after low temperature treatment; Figure 8 The figure shows the SOD activity measurement results of the plants before and after low-temperature treatment (D).

[0035] Figure 9 for SlBBX19 The results of the tolerance assessment of gene-overexpressing and knockout plants before and after high-temperature treatment are shown in the figure; among them, Figure 9 In the figure, A represents the results of malondialdehyde (MDA) content determination in plants before and after high-temperature treatment. Figure 9 In the figure, B represents the results of the relative electrical conductivity measurement of the plants before and after high temperature treatment; Figure 9 The "C" in the figure represents the results of POD activity measurement of plants before and after high temperature treatment; Figure 9 The figure shows the SOD activity measurement results of the plants before and after high temperature treatment, represented by the symbol D. Detailed Implementation

[0036] The present invention will be described in detail below with reference to specific embodiments, but should not be construed as limiting 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, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0037] Example 1:

[0038] one. SlBBX19 Tissue specificity of gene expression

[0039] 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 ripening stage; Br, color breaking stage; Br+3, three days after color breaking; Br+7, seven days after color breaking; Br+10, ten days after color breaking; Br+15, fifteen days after color breaking; seed, seed.

[0040] Tomato stress samples: Using the tomato variety 'Ailsa Craig' as the test material, plump seeds were selected, sterilized, and inoculated onto 20cm × 20cm 1 / 2 MS solid medium plates. The samples were then placed in an artificial climate chamber at 25℃, 70% relative humidity, and approximately 800 μmol / m² light intensity. -2 s -1 After culturing for 12 days under these conditions, the plates were transferred to a 4℃ / 42℃ light incubator, where the relative humidity was 70% and the light intensity was approximately 800 μmol / m². -2 s -1 Stress treatment was conducted under the specified conditions. Whole samples were taken from tomato seedlings subjected to low-temperature treatment for 0h, 1h, 3h, 6h, 12h, 24h, and Re (low-temperature treatment for 24h followed by recovery in a 25℃ light incubator for 1 day), and high-temperature treatment for 0h, 2h, 4h, 6h, 12h, 24h, and Re (high-temperature treatment for 24h followed by recovery in a 25℃ light incubator for 1 day). The 1 / 2 MS medium formulation was as follows: 2.23g MS powder was dissolved in 800mL of ultrapure water, 15g sucrose was added, and the volume was adjusted to 1000mL. The pH was adjusted to 5.8, 7.5g agar was added, and the medium was autoclaved at 121℃ for 21min.

[0041] Among them, 'Ailsa Craig' is abbreviated as AC. This material was propagated by Professor Zhan Xiangqiang's team at the Horticultural Crop Stress Biology Laboratory of Northwest A&F University.

[0042] MS powder: Phytotech, M519.

[0043] RNA was extracted from all samples using TRIzol (Tiangen) according to the kit instructions. First-strand cDNA was synthesized using the HiScript II 1st Strand cDNA Synthesis Kit, following the kit instructions. Quantitative primers were designed using Primer Premier 5.0 software, and gene expression was detected using a Power SYBR Green PCR MasterMix. Real-time quantitative RT-PCR was performed on a BIO-RAD CFX384 Touch.

[0044] qPCR reaction conditions: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 10 s, 40 cycles; 72℃ for 5 min.

[0045] qPCR reaction system, 15 μL: ddH2O 4.9 μL, SlBBX19-RT-F 0.3 μL, SlBBX19-RT-R 0.3 μL, Taq SYBR Green qPCR Premix (Universal) 7.5 μL, cDNA 2 μL.

[0046] Primer sequences:

[0047] The nucleotide sequence of SlBBX19-RT-F is shown in SEQ ID NO. 19:

[0048] 5'-GGCAGAGGATTGAGTTTCCG-3'.

[0049] The nucleotide sequence of SlBBX19-RT-R is shown in SEQ ID NO. 20:

[0050] 5'-CTCCCTGTTTTGGTTGGTGATT-3'.

[0051] II. Construction of overexpression vectors

[0052] 1. SlBBX19 Gene cloning

[0053] Download from the SGN tomato genome database SlBBX19 Based on the CDS sequence of the gene, specific primers OE-SlBBX19-F and OE-SlBBX19-R were designed. The primer sequences are shown in Table 1, SEQ ID NO. 1 to SEQ ID NO. 2. Amplification was performed. SlBBX19 The full-length cDNA sequence was obtained, and finally the cloned gene fragments were purified and recovered (e.g., Figure 3 (As shown in B in the diagram).

[0054] The SGN database can be found at https: / / solgenomics.net / .

[0055] SlBBX19 Gene accession number: Solyc01g110370.

[0056] SlBBX19 The size of the gene's CDS sequence is 726 bp.

[0057] PCR reaction conditions: 95℃ for 5 min; 95℃ for 30 s, 57℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 5 min.

[0058] PCR reaction system, 25 μL: ddH2O 7.5 μL, 2×Phanta Max Master Buffer 12.5 μL, OE-SlBBX19-F 1 μL, OE-SlBBX19-R 1 μL, dNTP 0.5 μL, template cDNA 2 μL, Phanta Max Super-Fidelity DNA Polymerase 0.5 μL.

[0059] 2. Constructing overexpression vectors:

[0060] (1) Use Xho I and Xba I. Restriction endonuclease linearization of the pHELLSgate2 vector. The pHELLSgate2 vector is as follows: Figure 3 As shown in A in the diagram.

[0061] (2) Homologous recombination is used to connect gene fragments and linearized vectors.

[0062] Reaction system: SlBBX19 Gene target fragment, 3 μL, linearized pHELLSgate2 Vector, 2 μL, LightNing DNA Assembly Mix Plus, 5 μL.

[0063] Reaction conditions: 50℃ for 60 min.

[0064] The ligation product was transformed into *E. coli* using a heat shock method. Colony PCR was performed using 35S-F and OE-SlBBX19-R primers for positive detection. The plasmid of the positive clone was extracted, sequenced using 35S-F primers, and sent to a company for sequencing. The correctly sequenced positive clone plasmid was named pHELLSgate2-SlBBX19. The detection results are as follows: Figure 3 As shown in C.

[0065] Table 1 SlBBX19 Gene cloning primers and detection primers

[0066]

[0067] III. Construction of Knockout Vector

[0068] 1. sgRNA sequence primer design

[0069] according to SlBBX19The genome sequence was obtained, and CRISPR RGEN Tools was used to design dual target sequences for sgRNA. The sgRNA1 and sgRNA2 sequences were selected as the target guide sequences for CRISPR-Cas9 knockout, and primer sequences were designed based on the target guide sequences.

[0070] The website address for CRISPR RGEN Tools is http: / / www.rgenome.net / .

[0071] The sgRNA1 and sgRNA2 sequences are shown in Table 2 as SEQ ID NO. 4 to SEQ ID NO. 5, and the primer sequences are shown in Table 2 as SEQ ID NO. 6 to SEQ ID NO. 7.

[0072] Table 2 sgRNA target sequences and sgRNA target primer sequences

[0073]

[0074] 2. Construction of CRISPR-Cas9 knockout vector

[0075] (1) Enzyme digestion of pBSE402 vector, enzyme digestion system: Bsa 0.5 μL of enzyme I, 10 μL of pBSE402 vector, 2 μL of Cut SmartBuffer, and 7.5 μL of ddH2O were added. The mixture was digested at 37℃ for 1 h. After electrophoresis, the linearized vector was recovered using the Tiangen DNA purification and recovery kit. The results are as follows: Figure 4 As shown in B in the diagram.

[0076] (2) PCR product amplification: Using the intermediate vector pCBC-DT1T2 as a template, PCR amplification was performed using SlBBX19-sgRNA-F and SlBBX19-sgRNA-R. The amplified fragments included sgRNA1 / U6-26t / U6-29p / sgRNA2, with a fragment size of 622bp.

[0077] Reaction system: ddH2O 7.5μL, 2×PhantaMax Master Buffer 12.5μL, SlBBX19-sgRNA-F 1μL, SlBBX19-sgRNA-R 1μL, dNTP 0.5μL, template pCBC-DT1T2 2μL, PhantaMaxSuper-Fidelity DNA Polymerase 0.5μL.

[0078] The reaction program was as follows: 95℃ for 5 min; 95℃ for 30 s, 57℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 5 min. The PCR products were detected by electrophoresis, and then recovered from the gel. The results are as follows: Figure 4 As shown in A in the diagram.

[0079] (3) sgRNA was ligated to the linearized pBSE402 vector. The reaction system consisted of 3 μL of the target sgRNA fragment, 2 μL of the linearized pBSE402 vector, and 5 μL of LightNing DNA Assembly Mix Plus. After ligation at 50℃ for 1 h, E. coli was transformed using the heat shock method. 100 mg / L kanamycin was used as the screening antibiotic. PCR was performed using primers U626-F and U629-R to detect positive results; the band size was 626 bp. The recombinant plasmid from the positive clone was extracted and sequenced using primers EDIT-3. The correctly sequenced recombinant plasmid was named SlBBX19-Cas9-pBSE402. The sequences of primers U626-F, U629-R, and EDIT-3 are shown in Table 3 as SEQ ID NO. 8~SEQ ID NO. 10. The detection results are as follows: Figure 4 As shown in C.

[0080] Table 3. Knockout vector detection primers and sequencing primers

[0081]

[0082] Four. SlBBX19 Obtaining gene overexpression plants and knockout plants:

[0083] 4.1 Tomato Seed Treatment

[0084] In a clean bench, soak tomato 'Ailsa Craig' AC seeds in sterile water for 30 minutes. After the seeds have absorbed water and swelled, discard the water. Add 75% alcohol (v / v) to an Erlenmeyer flask, shake for 30 seconds, then discard the alcohol. Wash the seeds with a 1:1 mixture of 84 disinfectant and sterile water for 8 minutes, shaking continuously. Discard the disinfectant solution, wash the tomato seeds twice with sterile ddH2O, and then inoculate the seeds onto 1 / 2 MS medium. Incubate the seeds in a climate chamber at 25℃ / 22℃ with 16h / 8h light for 7 days, at which point most seedlings will have fully expanded cotyledons.

[0085] 4.2 Preparation of explants

[0086] Sterile seedlings with fully expanded cotyledons were selected in a clean bench. Each cotyledon was cut into two explants with a scalpel. The obtained tomato cotyledon nodes were placed on a pre-culture medium and cultured at 25°C in the dark for 1 day to obtain tomato explants.

[0087] The pre-culture medium was KCMS medium, with the following formula: weigh 4.43 g / L MS powder, 30 g / L sucrose, adjust the pH to 5.8, add 7.5 g / L agar, and autoclave at 121℃ for 21 min.

[0088] MS powder: Phytotech, M524.

[0089] 4.3 Agrobacterium infection and transformation of tomatoes

[0090] (1) Take GV3101 Agrobacterium tumefaciens of pHELLSgate2-SlBBX19 or SlBBX19-Cas9-pBSE402 and streak it on LB solid medium containing 50 mg / L Rif and 50 mg / L Kan to activate it.

[0091] (2) Pick a single colony and add it to 15 mL of LB liquid medium containing 50 mg / L Rif and 50 mg / L Kan. Incubate at 28 °C with shaking until OD. 600 = Around 0.8.

[0092] (3) Centrifuge to collect bacteria, add an equal volume of Agrobacterium suspension, and add 100 mmol / L AS to prepare Agrobacterium infection solution.

[0093] (4) Place the obtained tomato explants into an empty glass dish, pour in Agrobacterium infection solution, shake continuously for 10 minutes, and finally transfer the tomato explants to a pre-culture medium and continue to culture in the dark for 2 days.

[0094] The LB liquid medium consisted of 5 g / L yeast extract, 5 g / L tryptone, and 10 g / L sodium chloride; the LB solid medium consisted of 5 g / L yeast extract, 5 g / L tryptone, 10 g / L sodium chloride, and 15 g / L agar.

[0095] Agrobacterium suspension: Weigh 4.43 g / L MS powder and 30 g / L sucrose, adjust the pH to 5.8, and autoclave at 121℃ for 21 min.

[0096] MS powder: Phytotech, M524.

[0097] 4.4 Screening and Culture

[0098] Tomato explants infected with Agrobacterium and co-cultured for 2 days were transferred to a selection medium. Approximately 30 explants were placed in each culture dish with the leaf surface facing up. The explants were cultured in a culture room at 25°C with 16h / 8h light for about 15 days until the callus tissue redifferentiated into adventitious buds.

[0099] The screening medium consisted of 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 termethin, with a pH of 5.8.

[0100] Among them, MS powder: Phytotech, M519.

[0101] For screening explants with the SlBBX19-Cas9-pBSE402 vector, 10% Basta antibiotic was used.

[0102] 4.5 Subculture

[0103] The selected tomato explants were transferred back to the subculture medium and cultured for about 15 days in an artificial climate chamber at 25°C with 16h / 8h light, until the callus tissue redifferentiated into adventitious buds.

[0104] The subculture medium consisted of 4.43 g / L MS powder, 30 g / L sucrose, 7.4 g / L agar, 0.2 mg / L zeatin ZR, 100 mg / L kanamycin (Kana) and 360 mg / L termethin, with a pH of 5.8.

[0105] Among them, MS powder: Phytotech, M519.

[0106] For the subculture of explants of the SlBBX19-Cas9-pBSE402 vector, 10% Basta antibiotic was used.

[0107] 4.6 Rooting Culture

[0108] When the adventitious buds grow to 2cm, cut them off from the base and transfer them to a rooting medium to induce rooting.

[0109] The rooting medium consisted of 4.43 g / L MS powder, 30 g / L sucrose, 7.4 g / L agar, 2 mg / L IBA, pH 5.8, 360 mg / L TMT + 50 mg / L kana.

[0110] Among them, MS powder: Phytotech, M519.

[0111] 4.7 Transplanting and Hardening Off

[0112] When the adventitious roots of the tomato seedlings have grown well, take out the tomato seedlings, clean the culture medium from the roots, transplant them into culture pots containing 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 a greenhouse for normal field management and used for propagation.

[0113] V. Identification of Transgenic Plants

[0114] 1. Identification of gain-of-function mutant plants

[0115] DNA was extracted from the leaves of the tomato plants to be tested and wild-type tomato plants using the CTAB method. The specific procedure was as follows:

[0116] (1) Take fresh leaves and place them in a 2mL centrifuge tube containing steel balls. After quick freezing with liquid nitrogen, grind them into powder evenly.

[0117] (2) Add 600 μL CTAB extract, shake to mix, and incubate in a water bath at 65°C for 30 min.

[0118] (3) Add 600 μL of chloroform to the fume hood, shake to mix, and centrifuge at 12000 rpm for 10 min.

[0119] (4) Carefully aspirate 400 μL of supernatant with a 1 mL pipette, transfer it to a new centrifuge tube, add an equal volume of isopropanol, mix well, and place in a -20℃ refrigerator for a sedimentation reaction of about 30 min.

[0120] (5) Centrifuge at 12000 rpm for 10 min and discard the supernatant.

[0121] (6) Add 1 mL of 75% ethanol by volume, shake to mix, centrifuge at 12000 rpm for 5 min, discard the supernatant, and place in a fume hood to evaporate the ethanol.

[0122] (7) Finally, add 100 μL of Tris-HCl solution, mix thoroughly, and store the DNA sample in a -20°C freezer. Tris-HCl: 10 μM, pH 8.0.

[0123] Identification of gain-of-function mutants using PCR:

[0124] 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.

[0125] The PCR program was as follows: 95℃ for 5 min; 95℃ for 30 s, 57℃ for 30 s, 72℃ for 1 min, 32 cycles; 72℃ for 5 min.

[0126] Based on the DNA detection results, DNA-positive plants were selected for RNA level detection. The specific procedures were as follows: RNA was extracted from all samples using TRIzol (Tiangen), following the kit instructions. First-strand cDNA was synthesized using the HiScript II 1st StrandcDNA Synthesis Kit (Vazyme), following the kit instructions. Quantitative primers were designed using Primer Premier 5.0 software, and gene expression was detected using Power SYBR Green PCR Master Mix (Applied Biosystems). Real-time quantitative RT-PCR (qPCR) was performed on a BIO-RAD CFX384 Touch. The quantitative primer sequences are shown in Table 4, SEQ ID NO. 11~SEQ ID NO. 14.

[0127] The RT-qPCR reaction system used was as follows: 7.5 μL of 2×ChamQ Universal SYBR qPCR Master Mix, 0.3 μL of 10 µM SlBBX19-qPCR-F, 0.3 μL of 10 µM SlBBX19-qPCR-R, 2 μL of cDNA, and 4.9 μL of ddH2O.

[0128] The RT-qPCR program was as follows: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 10 s (collect fluorescence), 40 cycles; 72℃ for 5 min. Tomato... SlACT7 The gene was used as an internal control gene, and each sample was tested in triplicate, using 2... -ΔΔCt Data calculations were performed using the fluorescence threshold method, and data analysis was conducted using Microsoft Excel 2019.

[0129] in, SlACT7 The gene's accession number is: Solyc11g005330.

[0130] Table 4 Quantitative Primers

[0131]

[0132] 2. Identification of loss-of-function mutant plants

[0133] DNA was extracted from leaves of transgenic and wild-type tomato plants using the CTAB method. Positive seedlings were detected using EDIT-3 and SlBBX19-SgRNA-R primers, and the DNA was analyzed using Primer Premier 5.0 software. SlBBX19Sequencing primers were designed approximately 300 bp before and after the target sites sgRNA1 and sgRNA2: SlBBX19-check1-F, SlBBX19-check1-R, and SlBBX19-check2-F, SlBBX19-check2-R. Using DNA from plants that tested positive for DNA as templates, fragments containing the sgRNA target sites were amplified. The PCR products were sent to a sequencing company for sequencing, and the sequences were compared with reference sequences to determine if mutations had occurred. The sequencing primer sequences are shown in Table 5, SEQ ID NO. 15~SEQ ID NO. 18.

[0134] Table 5 Sequencing Primers

[0135]

[0136] VI. Temperature stress tolerance assessment

[0137] Seeds from plump wild-type AC seedlings, as well as seeds from the two overexpression lines with the highest expression levels and two CRISPR / Cas9 knockout lines, were selected and germinated at 25℃ until they showed white sprouts. These seeds were then sown in 6.5cm diameter seedling cups. The culture conditions were: alternating light and dark conditions, with a daytime temperature of 25℃ and a light intensity of 20,000 lux for 16 hours, followed by a nighttime temperature of 20℃ for 8 hours. Seedlings were used for subsequent experimental treatments when they reached the five-leaf stage.

[0138] 1. Low-temperature treatment: When tomato seedlings reach the five-leaf stage, select healthy plants of similar size, with 15 plants from each line treated. These are randomly divided into two groups: one group is placed in an artificial climate chamber for constant temperature cultivation at 4℃, while the other group is placed at 25℃ as a control. Treatment at 4℃ lasts for 7 days, with a photoperiod of 16h / 8h, a light intensity of 20000 lux, and a relative humidity of 70%. Plant phenotypes are observed, and relevant physiological and biochemical indicators are measured for different lines, including relative conductivity, malondialdehyde (MDA) content, POD, and SOD activity.

[0139] 2. High-temperature treatment: Healthy plants of similar size from different strains were selected and placed in an artificial climate chamber for constant temperature incubation at 42℃ for 48 hours as the experimental group. Another group was placed at 25℃ as the control group. Both the experimental and control groups underwent alternating light and dark incubation simultaneously, with a photoperiod of 16h / 8h, a light intensity of 20000 lux, and a relative humidity controlled at approximately 50%. Plant phenotypes were observed, and relevant physiological and biochemical indicators of different strains were measured.

[0140] 3. Determination of Relative Conductivity: Add 10 ml of ddH2O to a 15 mL centrifuge tube, then add 10 blades with a diameter of 1 cm to the tube. Place the centrifuge tube on a shaker at 28°C and shake for 2 hours, then 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 formula for calculating relative conductivity (REL) is REL = EC1 / EC2 × 100%.

[0141] 4. Determination of malondialdehyde content and enzyme activity:

[0142] (1) Enzyme extraction:

[0143] 1) Weigh 0.1g of tomato leaves using a 0.01g balance and place them in a pre-cooled mortar. Remove as many veins as possible from the tomato leaves.

[0144] 2) Transfer the well-ground homogenate to a centrifuge tube and bring the phosphate buffer to a final volume of 1 mL; then place the centrifuge tube in an ice box and wait for centrifugation.

[0145] 3) Centrifuge at 4℃ and 12000 rpm for 20 minutes. The supernatant is the enzyme solution.

[0146] 4) After centrifugation, keep the product in an ice box and immediately measure the activity of antioxidant enzymes, etc.

[0147] (2) Determination of malondialdehyde content

[0148] 0.5% TBA (volume percentage): Weigh 5g of trichloroacetic acid and dissolve it in 100mL of water. Then weigh 0.5g of TBA, dissolve it in 10mL of 1M NaOH, and finally dilute to 100mL with the freshly prepared 5% trichloroacetic acid solution.

[0149] The method for preparing NaOH is to dissolve 4g in 100mL of water.

[0150] This reagent should be prepared and used immediately, stored at room temperature and protected from light. Add 150 μL of 0.5% TBA and 75 μL of enzyme solution sequentially to a 1.5 mL centrifuge tube, mix well, and incubate in a boiling water bath for 20 min. After 20 min, immediately place the tube in an ice-water bath to cool to room temperature. After cooling, centrifuge at 3000 rpm for 10 min, collect the supernatant, and measure the absorbance at 532 nm, 600 nm, and 450 nm using a UV spectrophotometer.

[0151] The calculation formula is:

[0152] MDA concentration (μmol / L) = 6.45 (OD) 532nm -OD 600nm -0.56×OD 450nm .

[0153] MDA content (μmol / g fresh weight) = MDA concentration (μmol / L) × extraction liquid volume (mL) / plant tissue fresh weight (g).

[0154] 5. Determination of POD activity

[0155] Prepare 50 mM (pH 7.0) phosphate buffer; add 1021 μL of 100 mM H2O2 (30% v / v) to a final volume of 100 mL, store at 4°C protected from light, and use immediately; add 0.4 mL of 100 mM guaiacol to a final volume of 100 mL, and store at 4°C. Initiate the reaction by adding 230 μL of 50 mM (pH 7.0) phosphate buffer, 30 μL of 100 mM guaiacol, 15 μL of enzyme solution, and 30 μL of 100 mM H2O2 sequentially. Measure the reaction at 470 nm using kinetic curves.

[0156] The calculation formula is:

[0157] POD activity (μmol g) -1 s -1 )=(A 470nm ×0.8) / (0.015×FW×26.8×60×3)=(Absorbance value×Extraction volume) / (Enzyme solution volume used in the determination×Fresh weight of sample×Extinction coefficient×Time).

[0158] 6. Determination of SOD activity

[0159] For SOD assay, place the reaction solution in a finger tube. Add the following to the sample tube sequentially: 160 μL of 0.05 mol / L phosphate buffer (pH 7.8), 30 μL of 130 mmol / L methionine (Met) solution, 30 μL of 750 μmol / L nitroblue tetrazolium solution, 30 μL of 100 μmol / L EDTA-Na2 solution, 20 μL of enzyme solution, and 30 μL of 20 μmol / L riboflavin solution. Add 180 μL of 0.05 mol / L phosphate buffer (pH 7.8) to the control tube, without adding enzyme solution. Add the other reagents in the same order as the sample tube. Measure the absorbance at 560 nm.

[0160] The formula for calculating SOD activity is:

[0161] 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 determination).

[0162] 7. Experimental Results

[0163] (1) SlBBX19 The gene is expressed in all tissues of the tomato.

[0164] like Figure 1 As shown, qRT-PCR detection revealed... SlBBX19 The gene is expressed in all tissues and organs of tomato AC (roots, stems, leaves, flowers, fruits, seeds, etc.).

[0165] (2) SlBBX19 The genes are significantly induced by low and high temperatures.

[0166] qRT-PCR was used to detect the effects of treatment at 4℃ (0h, 1h, 3h, 6h, 12h, 24h and after recovery) and treatment at 42℃ (0h, 2h, 4h, 6h, 12h, 24h and after recovery). SlBBX19 Gene expression levels. The results showed (see...) Figure 2 (A and B in the middle) SlBBX19 The gene showed a significant upward trend under low temperature stress, reaching its maximum value at 24 hours; SlBBX19 The gene showed a significant downregulation trend under high temperature stress, reaching its minimum at 2 hours. Therefore, it is speculated that the tomato... SlBBX19 Genes are involved in low-temperature and high-temperature stress responses.

[0167] (3) such as Figure 3 and Figure 4 As shown, the pHELLSgate2-SlBBX19 and SlBBX19-Cas9-pBSE402 vectors were successfully constructed.

[0168] (4) Successful creation of knockout and overexpression mutant plants

[0169] DNA was extracted and transgenic testing was performed, resulting in 11 transgenic positive lines (see...). Figure 5 (A and B in the original text). RNA was extracted from leaves of positive lines and detected by qRT-PCR. Quantitative fluorescence results showed that RNA was present in multiple transgenic lines. SlBBX19 The expression levels of these genes increased to varying degrees. OE-7 and OE-12 were selected for subsequent experiments and propagation.

[0170] Two knockout lines were obtained through sequencing analysis (see...). Figure 6 In comparisons A and B, it was found that the KO5 line had an 8bp deletion at the second target site. The KO5 line is a homozygous mutation, resulting in a frameshift in the target gene, premature appearance of the stop codon, and premature termination of protein translation. The KO41 line showed two editing methods at the second target site: one was a 1bp allele insertion, and the other was an 8bp allele deletion. Both resulted in a frameshift in the target gene, premature appearance of the stop codon, and premature termination of protein translation. Both knockout lines can be used for subsequent experiments and propagation.

[0171] (5) Knockout SlBBX19 Genes significantly improve the low-temperature tolerance of tomato plants.

[0172] Phenotypic observation revealed (see...) Figure 7 (A) After 7 days of low-temperature treatment, the overexpressing plants showed severe wilting and yellowing, while the knockout plants grew well and exhibited significant resistance. Malondialdehyde (MDA) content and relative conductivity can be used to assess the degree of damage to plants under abiotic stress. Therefore, after low-temperature treatment, the MDA content and relative conductivity of the overexpressing plants were significantly higher than those of the knockout plants, indicating that the overexpressing plants suffered more severe damage. Enzyme activity assays revealed that the POD and SOD activities of the knockout plants were significantly higher than those of the wild-type and overexpressing plants after low-temperature treatment, indicating that the knockout lines had a stronger reactive oxygen species (ROS) scavenging capacity, resulting in less ROS damage (see [reference needed]). Figure 8 In summary, knockout SlBBX19 Genetic modification significantly improved the low-temperature tolerance of tomatoes.

[0173] (6) Knockout SlBBX19 Genes have improved the heat resistance of tomatoes.

[0174] Phenotypic observation revealed (see...) Figure 7 (B) After 48 hours of high-temperature treatment, the overexpressing plants showed severe wilting, drying, and yellowing, while the knockout plants grew well. Furthermore, the malondialdehyde (MDA) content and relative conductivity of the overexpressing plants were significantly higher than those of the wild-type and knockout plants after high-temperature treatment; the POD and SOD activities of the overexpressing plants were significantly lower than those of the wild-type and knockout plants (see section B). Figure 9 The above explains how to remove... SlBBX19 Genetic modification significantly improved the heat resistance of tomatoes.

[0175] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0176] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. SlBBX19 The use of gene expression inhibitors in any of the following, characterized in that, a. Application in improving the tolerance of tomatoes to temperature stress; b. Application in cultivating tomatoes with high tolerance to temperature stress; The SlBBX19 The gene accession number is Solyc01g110370; The tolerance to temperature stress includes: tolerance to high temperatures and / or tolerance to low temperatures. The SlBBX19 Gene expression inhibitors include: targeted knockout SlBBX19 CRISPR / Cas9 vectors for genes; Targeted knockout SlBBX19 The CRISPR / Cas9 vector of the gene contains a target SlBBX19 The sgRNA sequence of the 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.

2. A method for improving the tolerance of tomatoes to temperature stress, characterized in that, include: By inhibiting the as described in claim 1 in tomatoes SlBBX19 Gene expression to improve tomato tolerance to temperature stress; The SlBBX19 The gene accession number is Solyc01g110370; Inhibit the above-mentioned in tomatoes SlBBX19 Methods for gene expression include: using CRISPR / Cas9 editing to specifically knock out the gene described in tomatoes. SlBBX19 Gene; The tolerance to temperature stress includes tolerance to high temperatures and / or tolerance to low temperatures.

3. The method as described in claim 2, characterized in that, The CRISPR / Cas9 editing method uses vectors that include targeted knockout. SlBBX19 CRISPR / Cas9 vectors for genes; Targeted knockout SlBBX19 The CRISPR / Cas9 vector of the gene contains a target SlBBX19 The sgRNA sequence of the gene; The sgRNA sequence includes a nucleotide sequence as shown in SEQ ID NO. 4 or a nucleotide sequence as shown in SEQ ID NO. 5.

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