Application of tomato SlKUF7 protein and coding gene thereof in heat-resistant genetic improvement of fruits

Abstract

The invention discloses application of tomato SlKUF7 protein and a coding gene thereof in heat-resistant genetic improvement of fruits, and belongs to the technical field of genetic engineering. According to the invention, the SlKUF7 protein related to the heat resistance of tomato fruits is screened, the amino acid sequence of the SlKUF7 protein is shown as SEQ ID NO.2, the nucleotide sequence of the coding gene is shown as SEQ ID NO.1, and a tomato plant over-expressing the tomato SlKUF7 protein coding gene is constructed. A high-temperature stress experiment result shows that compared with a non-transgenic line, the tomato line overexpressing the tomato SlKUF7 protein coding gene has the advantages that the fruit heat resistance is enhanced, the fruit hardness is obviously improved, and the fruit abscisic acid content is obviously increased. The results show that the tomato SlKUF7 protein positively regulates the heat resistance of the fruits, and the coding gene of the tomato SlKUF7 protein can be used as a target gene for molecular genetic breeding of tomato varieties with heat resistance of the fruits, and has important significance for breeding of tomato varieties cultivated by facilities.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the tomato SlKUF7 protein and its encoding gene in the genetic improvement of fruit heat resistance. Background Technology

[0002] Tomato (Solanum lycopersicum) is an important horticultural economic crop worldwide, widely used in fresh consumption, processing, and nutritional health food. Its fruit is rich in antioxidants such as vitamin C and lycopene, and has considerable market value. However, tomato fruit is particularly sensitive to high temperatures, especially under greenhouse cultivation conditions. When the ambient temperature exceeds 35°C, the appearance of the fruit (if the skin is intact) will significantly decline, directly affecting fruit quality and economic benefits. With the intensification of global warming, high temperature stress has become one of the key factors restricting tomato production, especially greenhouse cultivation, and sustainable development. Therefore, breeding heat-resistant tomato varieties is of great significance for maintaining the safety of horticultural crop production and improving agricultural economic benefits. At present, research on plant heat resistance mainly focuses on three aspects: (1) the discovery of heat resistance-related genes: reported genes include heat shock proteins (such as HSP70 / 90), transcription factors (such as HSFA1 and HSFB1), and antioxidant enzymes (such as APX and SOD) genes. These genes enhance heat resistance by regulating protein homeostasis and oxidative stress response; (2) Location of heat resistance gene loci: Through population genetic analysis, multiple quantitative trait loci (QTLs) related to heat resistance were found, but their specific functional genes have not been fully analyzed; (3) Heat resistance induced by exogenous substances: Exogenous application of melatonin or salicylic acid can alleviate heat damage by activating the antioxidant system, but this method is costly and the effect is unstable.

[0003] Although previous research has made some progress in enhancing plant heat tolerance, the limited effectiveness of individual genes involved in basal stress responses and the complex quantitative trait of heat tolerance means that there is still a lack of research and utilization of genes targeting key regulatory nodes of fruit-specific heat tolerance. F-box proteins, as core components of the ubiquitin-proteasome system (UPS), can specifically degrade substrate proteins and regulate various plant hormone (such as abscisic acid) signaling pathways, making them important regulatory factors in plant abiotic stress responses. Studies have shown that F-box proteins can affect heat tolerance by regulating the stability of heat shock transcription factors (HSFs), but their function in fruit heat tolerance has not been fully utilized. To overcome the limitations of existing technologies (functional redundancy of basal heat tolerance genes and unsatisfactory application effects), this invention aims to improve the heat tolerance of tomato fruits through transgenic technology using the tomato F-box protein SlKUF7 and its encoding gene. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the tomato SlKUF7 protein and its encoding gene in the genetic improvement of fruit heat resistance, in order to solve the problems existing in the prior art. This invention constructs tomato plants that overexpress the tomato SlKUF7 protein encoding gene. Compared with non-transgenic lines, the tomato lines that overexpress the tomato SlKUF7 protein encoding gene have enhanced fruit heat resistance, significantly improved fruit firmness, and significantly increased abscisic acid content. The tomato SlKUF7 protein encoding gene can be used as a target gene for the molecular genetic breeding of heat-resistant tomato varieties.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides an application of tomato SlKUF7 protein in any of the following:

[0007] (1) Application in regulating the heat resistance of tomato fruits;

[0008] (2) Application in the cultivation of heat-resistant transgenic tomatoes;

[0009] (3) Application in the preparation of products that improve the heat resistance of tomato fruits;

[0010] The amino acid sequence of the tomato SlKUF7 protein is shown in SEQ ID NO.2.

[0011] Furthermore, the expression level of the tomato SlKUF7 protein was upregulated in tomatoes, thereby improving the heat resistance of tomato fruits.

[0012] The present invention also provides an application of the gene encoding the above-mentioned tomato SlKUF7 protein in any of the following:

[0013] (1) Application in regulating the heat resistance of tomato fruits;

[0014] (2) Application in the cultivation of heat-resistant transgenic tomatoes;

[0015] (3) Application in the preparation of products that improve the heat resistance of tomato fruits;

[0016] The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0017] Furthermore, the expression level of the encoded gene was upregulated in tomatoes, thereby improving the heat resistance of tomato fruits.

[0018] The present invention also provides an application of a gene overexpression vector, wherein the overexpression vector contains the gene encoding the tomato SlKUF7 protein;

[0019] The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1;

[0020] The application is any one of the following:

[0021] (1) Application in regulating the heat resistance of tomato fruits;

[0022] (2) Application in the cultivation of heat-resistant transgenic tomatoes;

[0023] (3) Application in the preparation of products that improve the heat resistance of tomato fruits.

[0024] The present invention also provides the use of engineered bacteria comprising the above-described overexpression vector in any of the following:

[0025] (1) Application in regulating the heat resistance of tomato fruits;

[0026] (2) Application in the cultivation of heat-resistant transgenic tomatoes;

[0027] (3) Application in the preparation of products that improve the heat resistance of tomato fruits.

[0028] Furthermore, the indicators of the fruit's heat resistance include fruit firmness.

[0029] The present invention also provides a method for improving the heat resistance of tomato fruit, comprising the step of upregulating the expression level of the gene encoding the tomato SlKUF7 protein in tomatoes to improve the heat resistance of the tomato fruit;

[0030] The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0031] This invention also provides a breeding method for transgenic tomatoes with heat-resistant fruit, comprising the following steps:

[0032] The gene encoding the tomato SlKUF7 protein was overexpressed in tomato cells, the tomato cells were then cultured, and tomatoes were regenerated using the tomato cells to obtain heat-resistant transgenic tomatoes.

[0033] The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0034] Furthermore, the indicators of the fruit's heat resistance include fruit firmness.

[0035] The present invention discloses the following technical effects:

[0036] This invention screened the SlKUF7 protein, which is associated with heat tolerance in tomato fruit. Its amino acid sequence is shown in SEQ ID NO. 2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 1. Tomato plants overexpressing the SlKUF7 protein encoding gene were constructed. High-temperature stress experiments showed that, compared to non-transgenic lines, tomato lines overexpressing the SlKUF7 protein encoding gene exhibited enhanced fruit heat tolerance, significantly increased fruit firmness, and significantly higher abscisic acid content. These results indicate that the tomato SlKUF7 protein positively regulates fruit heat tolerance, and its encoding gene can be used as a target gene for molecular genetic breeding of heat-tolerant tomato varieties, which is of great significance for the breeding of greenhouse-grown tomato varieties. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 The images show the detection results of SlKUF7 gene overexpression tomato plants. A is a schematic diagram of the construction of the SlKUF7 gene overexpression vector pCambia13000-SlKUF7-HA; B shows the results of SlKUF7 gene transcription level detection in wild-type and SlKUF7 gene overexpression tomato fruits; C shows the SlKUF7 protein expression level detection in wild-type and SlKUF7 gene overexpression tomato fruits; WT represents wild-type, and OE-14 and OE-19 represent SlKUF7 gene overexpression tomato lines.

[0039] Figure 2 The figures show the statistical results of fruit phenotype and fruit firmness of wild-type and SlKUF7 gene-overexpressing tomatoes; where A represents the fruit phenotype of wild-type and SlKUF7 gene-overexpressing tomatoes after high-temperature treatment, with a scale bar of 2 cm; B represents the fruit firmness measurement of wild-type and SlKUF7 gene-overexpressing tomatoes after high-temperature treatment; WT represents wild-type, and OE-14 and OE-19 represent SlKUF7 gene-overexpressing tomato lines.

[0040] Figure 3 The image shows the abscisic acid content in wild-type and SlKUF7 gene-overexpressing tomato fruits; WT represents wild-type; OE-14 and OE-19 represent SlKUF7 gene-overexpressing tomato lines. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0045] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0046] In this invention, Kan represents kanamycin, Rif represents rifampin, Gen represents gentamicin, and AS represents acetylsyringone.

[0047] The amino acid sequence of the tomato SlKUF7 protein of the present invention is shown in SEQ ID NO.2, and the nucleotide sequence of its encoding gene (SlKUF7 gene) is shown in SEQ ID NO.1.

[0048] SEQ ID NO.1:

[0049]

[0050] SEQ ID NO.2:

[0051] MNNSTTACAAVNRRKILHHRRHHQSLIPGLPDDVAQICLNQVHPSTLFSVCHSWRKLIYSPSFPFLSLYALLLSTNQVEFACFDPISSRWHLLPPPPPDPPLRILVKHPSFISRNLPIQSVTVSGNLILLAATADQFLPAISRPLIFNPTVKKWTHGPRLNAPRRWCAAGALGNSVYVASGVGSHYNLDVAR SVIKWDLDNSSGPEFDNRAHYSARYRNERKMRWKWEEMSGLRDGKFSREAIEAIGWKGKLCMVNVKGDAAKQGIIYNVETDTWHDMPEGMLSGWRGPVAAMAEEILYTVDESKGVLRRYDEERDVWVEILEYDMLKGAEHVVADGGRVCVVGGGGGSGIVVVDVAVETPRLVMVETPVGFQVLNIHVLPRMSH.

[0052] Example 1

[0053] 1. Materials and Methods

[0054] 1.1 Experimental Materials

[0055] The plant material used in this invention is the tomato variety (Solanum lycopersicum) Ailsa Craig, abbreviated as AC tomato.

[0056] Strains: Escherichia coli DH5α competent cells, Agrobacterium GV3101 competent cells.

[0057] Vector: The overexpression vector is the pCambia1300-MCS-3×HA plasmid vector, which is a modified version of the pCambia1300 vector.

[0058] 1.2 Cloning and Overexpression Vector Construction of SlKUF7 Gene

[0059] 1.2.1 Total RNA Extraction

[0060] RNA was extracted from leaves using the Trizol method, and the specific steps are as follows:

[0061] 1) Weigh 0.1 g of tomato leaf tissue and grind it into powder quickly in a mortar containing liquid nitrogen using a grinding stick. Place the ground powder into a 1.5 mL RNase-free centrifuge tube and add 1000 μL of TransZol Up extraction solution (Beijing TransGen, catalog number: ER501-01V2-01) to the RNase-free centrifuge tube. Place on ice for 5 min.

[0062] 2) Add 200 μL of chloroform, shake upside down for 15 seconds, and store in a refrigerator at -20°C for 3 minutes;

[0063] 3) Remove the sample and place it in a centrifuge. Centrifuge at 4°C and 12,000 rpm for 20 min.

[0064] 4) Remove the centrifuged sample, aspirate 500 μL of the supernatant into a new RNase-free centrifuge tube, add 500 μL of isopropanol, shake gently up and down 8 times, and store at -20℃ for 20 min.

[0065] 5) Take out the sample and put it into a centrifuge. Centrifuge at 4℃ and 12000 rpm for 10 min. After centrifugation, discard the supernatant and leave a white flocculent precipitate.

[0066] 6) Add 1000 μL of 75% ethanol (prepared with DEPC water), gently blow with a pipette tip until the precipitate disappears, then centrifuge;

[0067] 7) Place the sample in a centrifuge and centrifuge at 4℃ and 12000 rpm for 10 min. Discard the supernatant after centrifugation.

[0068] 8) Add 1000 μL of 75% ethanol (prepared with DEPC water), gently blow with a pipette tip until the precipitate disappears, then centrifuge;

[0069] 9) Place the sample in a centrifuge and centrifuge at 4℃ and 12000 rpm for 10 min. Discard the supernatant after centrifugation.

[0070] 10) Place the washed sample in the laminar flow hood and air dry;

[0071] 11) Add 30 μL of DEPC water, detect the RNA concentration, and store at -80℃.

[0072] 1.2.2 Reverse transcription to synthesize cDNA

[0073] Perform cDNA synthesis according to the instructions of the Novozymes HiScriptIII 1st Stand cDNA Synthesis Kit (+gDNAwiper) (Catalog No.: R312-01). The cDNA product can be used immediately for PCR reaction or stored in a -20°C freezer.

[0074] 1.2.3 PCR amplification of the target gene

[0075] 1) Primers were designed using Primer Premier 5 software.

[0076] The primer sequences for cloning the SlKUF7 gene are shown in SEQ ID NO.3-4.

[0077] SlKUF7-F: ACGGGGGACGAGCTCGGTACCATGAACAACTCCACCACCGC, SEQ ID NO.3;

[0078] SlKUF7-R: CGCGTACGAGATCTGGTCGACATGGCTCATCCTAGGCAAAAC, SEQ ID NO. 4.

[0079] 2) Perform PCR amplification reaction

[0080] According to Tables 1 and 2, a 50 µL PCR amplification reaction was performed. The cDNA from tomato leaves was used as a template and the gene was amplified using the high-fidelity DNA polymerase TransStart FastPfu Fly PCR SuperMix (catalog number: AS231). The PCR amplification products were detected by agarose gel electrophoresis. The amplified band size was observed to be about 1200 bp under UV light, which means that the SlKUF7 gene fragment was cloned.

[0081] Table 1 PCR amplification system

[0082] PCR reaction system Usage TransStart FastPfu Fly PCR SuperMix 25 µL SlKUF7-F 1 µL SlKUF7-R 1 µL cDNA template 1 µL <![CDATA[ddH2O]]> 22 µL

[0083] Table 2 PCR reaction procedure

[0084]

[0085] 1.2.4 Agarose gel electrophoresis and gel recovery of target gene fragments

[0086] The target gene fragment was recovered by gel extraction using the FastPure Gel DNA Extraction Mini Kit (Novizan, catalog number: DC301). The gel extraction product was then stored at -20°C.

[0087] 1.2.5 Construction of Overexpression Vectors

[0088] 1) Linearization of expression vectors

[0089] The DH5α *E. coli* strain containing the pCambia1300-MCS-3×HA plasmid vector, stored at -80℃, was removed. Single colonies were streaked and placed in LB broth. The strain was then incubated overnight at 37℃ and 200 rpm in a shaker. The pCambia1300-MCS-3×HA plasmid was extracted. The plasmid vector was double-digested using NEB-brand KpnI-HF and SalI-HF restriction endonucleases in a 50 µL reaction volume as shown in Table 3. The double digestion reaction was performed at 37℃ for 1 h. Agarose gel electrophoresis was used to verify whether the vector was cleaved. The linearized vector fragment was obtained using the gel recovery method described above, and its concentration was measured and stored at -20℃ for later use.

[0090] Table 3. Double enzyme digestion reaction system

[0091] Components Usage 10×Buffer 5 µL KpnI 1 µL SalI 1 µL pCambia1300-MCS-3×HA vector 16 µL <![CDATA[ddH2O]]> 27 µL

[0092] 2) Homologous recombination linkage

[0093] The sample volumes of the linearized vector and target gene fragment obtained from gel extraction were calculated based on their concentrations and base counts. Ligation was performed using the ClonExpress® II One Step Cloning Kit (Novizan, catalog number: C112-01) at a 1:2 ratio of linearized vector to target gene fragment. The homologous recombination reaction system was prepared on ice according to Table 4. The prepared 10 µL homologous recombination system was incubated at 37°C for 30 min in a PCR instrument. The resulting homologous recombination product was then transformed into *E. coli*.

[0094] Table 4 Homologous recombination reaction system

[0095] Components Usage 5×CEⅡ Buffer 2 µL Exnase II 1 µL Linearized pCambia1300-MCS-3×HA vector 1 µL SlKUF7 gene fragment 1 µL <![CDATA[ddH2O]]> 5 µL

[0096] 3) Transformation of Escherichia coli

[0097] ① Remove DH5α competent cells (Weidi Bio, DL1001) from the -80℃ freezer and place them on ice for later use. Add competent cells to a sterile 1.5 mL centrifuge tube, and add 10 µL of the ligated recombinant DNA product to the 1.5 mL sterile centrifuge tube containing competent cells (operate on ice), and place on ice for 30 min;

[0098] ②After the ice bath, place it in a water bath and heat shock it at 42°C for 1 minute, then place it on ice for 3 minutes;

[0099] ③ Perform the operation in a clean bench, add 800 µL of liquid LB medium to a 1.5 mL centrifuge tube, seal the 1.5 mL centrifuge tube with sealing film, place it in a shaker, and treat at 37℃ and 200 rpm for 1 h;

[0100] ④ Remove the 1.5 mL centrifuge tube from the shaker, place the centrifuge tube in the centrifuge, and centrifuge at 25℃ and 5000 rpm for 5 min;

[0101] ⑤ After centrifugation, remove part of the supernatant in a clean bench, leaving about 100 µL of supernatant in the centrifuge tube. Mix the supernatant with the precipitate using a sterilizing pipette tip, and spread the bacterial culture onto LB solid medium (with Kan added) that has been poured into a petri dish in advance. Incubate overnight at 37°C.

[0102] ⑥ After the colonies have grown, pick a single colony and put it into LB liquid medium (add Kan to LB liquid medium), place it in a shaker, and incubate overnight at 37°C and 200 rpm;

[0103] ⑦ Using bacterial culture as a template, the detection primers shown in SEQ ID NO.5-6 and TransFast Taq PCR SuperMix (TransFast, catalog number: AS102) were used to perform the detection according to the PCR reaction system and procedure in Tables 5 and 6;

[0104] 1300-F: TTGGAGAGAACACGGGGGAC, SEQ ID NO.5;

[0105] 1300-R: CGATGATACGAACGAAAGCT, SEQ ID NO. 6.

[0106] Table 5 Taq enzyme PCR amplification system

[0107] PCR reaction system Usage TransFast Taq PCR SuperMix 10 µL 1300-F 1 µL 1300-R 1 µL Bacterial template 1 µL <![CDATA[ddH2O]]> 7 µL

[0108] Table 6 Taq enzyme PCR reaction procedure

[0109]

[0110] ⑧ After the reaction is complete, the PCR products are detected by agarose gel electrophoresis;

[0111] ⑨ Select bacterial cultures that showed positive results by agarose gel electrophoresis to extract plasmids.

[0112] 4) Plasmid extraction

[0113] Plasmid extraction was performed using the FastPure Plasmid Mini Kit (Novozymes, catalog number: DC201-01) to obtain the recombinant plasmid pCambia1300-SlKUF7-3×HA, which was then sent to the company for sequencing. Recombinant plasmids that successfully aligned with the sequencing results were used for subsequent experiments.

[0114] 1.2.6 Transformation of Agrobacterium with recombinant plasmids

[0115] The recombinant plasmid pCambia1300-SlKUF7-3×HA, which had the correct sequencing results, was transformed into GV3101 Agrobacterium competent cells (Weidi Biotechnology, catalog number: AC1001) using the freeze-thaw method. The operation steps are as follows:

[0116] 1) Take Agrobacterium competent cells GV3101 out of the -80℃ freezer, place them on pre-made ice to thaw, and then aspirate them into a sterile 1.5 mL centrifuge tube;

[0117] 2) Ice operation: Add 1 µL of plasmid to Agrobacterium competent cells GV3101, and immediately place the centrifuge tube in ice for 30 min.

[0118] 3) Immediately after the ice bath, place the centrifuge tubes in liquid nitrogen for 5 minutes to freeze quickly;

[0119] 4) After quick-freezing, place the centrifuge tubes in a water bath and incubate at 37°C for 5 minutes;

[0120] 5) After the water bath, place the centrifuge tubes on ice for 5 minutes.

[0121] 6) After the ice bath, remove the centrifuge tube, add 800 µL of liquid LB medium to the centrifuge tube in the clean bench, seal the centrifuge tube with sealing film, and place the centrifuge tube in a shaker and shake at 28℃ for 3-4 h.

[0122] 7) Place the centrifuge tubes in a centrifuge at 25°C and 5000 rpm for 5 minutes;

[0123] 8) After centrifugation, remove part of the supernatant in the clean bench with a pipette tip. Mix the remaining 100µL of supernatant with a pipette tip, spread the liquid onto the pre-pouring LB solid medium (containing Kan, Rif and Gen), seal it with sealing film and place it in an incubator. Incubate at 28℃ for about 2 days.

[0124] 9) After the bacteria have grown on the petri dish, pick a single colony and put it into LB liquid medium (containing Kan, Rif and Gen), and place it in a shaker at 28°C for inoculation;

[0125] 10) After shaking the bacterial suspension, perform Agrobacterium PCR detection according to the above Escherichia coli bacterial suspension PCR detection method. Detect the bacterial suspension using agarose gel electrophoresis. Preserve the bacterial suspension corresponding to the positive band at -80℃ for subsequent research.

[0126] 1.3 Genetic transformation of tomatoes and identification of transgenic positive plants

[0127] 1.3.1 Agrobacterium-mediated genetic transformation

[0128] 1) Culture medium preparation

[0129] Prepare the culture medium required for this invention according to the formula listed in Table 7 for subsequent tissue culture.

[0130] Table 7 Culture medium composition

[0131] Culture medium name Culture medium components MS liquid culture medium MS + 3% sucrose MS solid culture medium MS + 3% sucrose + 0.3% plant gel Germination medium MS + 3% sucrose + 0.3% plant gel Pre-culture medium MS + 3% sucrose + 0.3% plant gel + 1 mg / L zeatin + 0.1 mg / L auxin co-culture medium MS + 3% sucrose + 0.3% plant gel + 100 µL AS + 2 mg / L zeatin + 0.1 mg / L auxin Screening culture medium MS + 3% sucrose + 0.3% plant gel + 2 mg / L zeatin + 0.1 mg / L auxin + 10 mg / L hygromycin + 400 mg / L termethin Bud elongation medium MS + 3% sucrose + 0.3% plant gel + 0.2 mg / L zeatin + 0.1 mg / L auxin + 10 mg / L hygromycin + 400 mg / L termethin Rooting medium MS + 3% sucrose + 0.3% plant gel + 1 mg / L indolebutyric acid + 10 mg / L hygromycin + 400 mg / L termethin

[0132] 2) Aseptic seedling culture

[0133] ① Take 40 vigorous and plump AC tomato seeds and place them in a beaker. Add distilled water to cover the seeds and soak overnight at room temperature.

[0134] ② In the clean bench, rinse the soaked seeds with sterile distilled water to remove impurities from the seeds;

[0135] ③ Immerse the seeds in 75% alcohol for 2 minutes;

[0136] ④ Rinse the seeds soaked in 75% alcohol three times with sterile distilled water to remove any residual alcohol from the seeds;

[0137] ⑤ Add a sodium hypochlorite solution with an effective chloride ion concentration of 2% to the seeds and soak the seeds for 15 minutes. At this time, the seeds turn white and some seeds begin to float.

[0138] ⑥ Rinse the seeds soaked in sodium hypochlorite three times with sterile distilled water;

[0139] ⑦ In a clean bench, spread the seeds evenly on the germination medium that has been pre-packaged in culture bottles, trying to avoid overlapping the seeds. Cultivate in the dark for 3 days. After the seeds germinate, place them under light for one week. At this time, the cotyledons of the tomato seedlings will unfold.

[0140] 3) Cotyledon pre-culture

[0141] When the cotyledons of the tomato seedlings unfold, use a sterile scalpel in a clean bench to cut off both ends of the cotyledons, leaving a rectangular leaf about 0.5 cm long in the middle. Carefully pick up the cut leaf with tweezers and place it in the pre-culture medium, making sure that the cut end of the cotyledon is in contact with the pre-culture medium as much as possible. Incubate the cut cotyledons in the dark for 2 days for subsequent infection operations.

[0142] 4) Agrobacterium infection

[0143] ① Picking bacteria: Add LB liquid medium (containing 50 mg / L Kan, 50 mg / L Rif and 50 mg / L Gen) to a 50 mL conical flask, pick a single colony of Agrobacterium and place it in the conical flask, then incubate overnight at 28°C and 200 rpm.

[0144] ② Transplantation bacteria: Add LB liquid medium (containing 50 mg / L Kan, 50 mg / L Rif, 50 mg / L Gen and 100 µM AS) and overnight cultured bacterial solution to a 250 mL Erlenmeyer flask at a ratio of 1:1000. Incubate at 28℃ and 200 rpm in a shaker. Measure the OD of the bacterial solution using a spectrophotometer. 600 Stop culturing when the concentration is around 0.5.

[0145] ③Collect bacteria: OD 600 The bacterial culture, with a volume of approximately 0.5, was placed in a centrifuge and centrifuged at 25°C, 5000 rpm for 5 min. The supernatant was discarded after centrifugation. The collected bacterial cells were washed once with MS liquid medium, centrifuged again at 25°C, 5000 rpm for 5 min, and the cells were resuspended in MS liquid medium. The OD of the resuspended bacterial culture was adjusted to [value missing]. 600 The concentration is 0.5. At this point, AS is added to the MS liquid medium containing bacterial solution for subsequent infection operations.

[0146] ④ Infection: In a clean bench, use tweezers to remove the tomato cotyledons from the pre-medium and immerse them in the bacterial solution prepared in the previous step. Shake continuously during immersion to prevent bacterial sedimentation. Immersion time is 15 minutes. After infection, carefully remove the infected tomato cotyledons with tweezers and place them on dry, sterile filter paper to absorb the bacterial solution. Then carefully transfer the tomato cotyledons to the co-medium and incubate in the dark at room temperature for 2 days.

[0147] 5) Screening for resistant buds

[0148] After co-culturing tomato cotyledons with Agrobacterium, the tomato cotyledons were transferred to a selection medium to screen for resistant shoots. Under room temperature conditions, with 16 hours of light and 8 hours of darkness, resistant shoots could grow in about 20 days.

[0149] 6) Bud elongation culture

[0150] After resistant shoots emerge from the selection medium, the shoots are carefully transferred to the shoot elongation medium and cultured at room temperature for 16 hours in light and 8 hours in darkness for about 14 days.

[0151] 7) Bud rooting culture

[0152] When the resistant shoots grow to 1-2 cm, carefully remove them from the shoot elongation medium and transfer them to the rooting medium to induce root differentiation. Once the root system is well-developed, subsequent operations can be carried out.

[0153] 8) Seedling transplanting

[0154] When the seedlings in the culture bottle have developed a well-developed root system, open the culture bottle and place it under light conditions. After 1-2 days, carefully remove the seedlings from the culture bottle and clean the culture medium from the roots of the seedlings with clean water. Plant the seedlings in a plastic pot filled with soil. Before transplanting, cover the seedlings with plastic bags and place them in a light incubator. When the seedlings have developed a well-developed root system and thick stems, transfer them to a greenhouse for transplanting.

[0155] 1.3.2 Identification of transgenic plants

[0156] 1) Genomic DNA extraction

[0157] DNA was extracted from the leaves of wild-type and transgenic tomato plants using the FastPure Plant DNA Isolation Mini Kit (Novizan, DC104-01) and then preserved.

[0158] 2) PCR identification of transgenic plants

[0159] Using genomic DNA from transgenic plants as a template, the pCambia1300-SlKUF7-3×HA recombinant plasmid was used as a positive control, and wild-type plant genomic DNA was used as a negative control. PCR detection was performed according to the primers (SEQ ID NO. 5-6), system (Table 5), and procedure (Table 6) for the bacterial culture PCR reaction described above. The PCR products of the transgenic plants were analyzed by agarose gel electrophoresis, and samples with PCR products of approximately 1200 bp were considered positive.

[0160] 3) qRT-PCR analysis of transgenic plants

[0161] Wild-type and PCR-positive transgenic tomato plants were transplanted into a greenhouse and harvested when the fruit reached the green-ripe stage. Tomato peels were collected, cut into small pieces, and immediately frozen in liquid nitrogen, then stored at -80°C for later use. Total RNA was extracted from the fruits using the same method described above, and cDNA was synthesized via reverse transcription. Using cDNA from both wild-type and transgenic tomato fruits as templates, the transcriptional level of the target gene was analyzed using the ChamQ SYBR qPCR Master Mix qRT-PCR kit (Novizan, catalog number: Q311).

[0162] ① Use Primer Premier 5 software to design qRT-PCR detection primers as shown in SEQ ID NO.7-10.

[0163] qSlKUF7-F: TGATATGCTTAAAGGCGCGGAAC, SEQ ID NO.7;

[0164] qSlKUF7-R: ACCAACCACGCAAACTCTACCAC, SEQ ID NO.8;

[0165] qActin-F: ACAACTTTCCAACAAGGGAAGAT, SEQ ID NO.9;

[0166] qActin-R: TGTATGTTGCTATTCAGGCTGTG, SEQ ID NO. 10.

[0167] ② Perform qRT-PCR amplification according to the reaction system in Table 8 and the reaction procedure in Table 9, setting Actin as the internal reference gene, and using 2 -△△Ct The relative expression level of the SlKUF7 gene was calculated. The qRT-PCR reaction was performed in triplicate, and the data were presented in a bar chart using the mean and standard deviation of the three datasets. The significance analysis between SlKUF7 overexpressing tomato fruits and wild-type was performed using a two-tailed Student's t-test (* represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001).

[0168] Table 8 qRT-PCR reaction system

[0169] Components Usage ChamQ SYBR qPCR Master Mix 10 µL Primer F 0.4 µL Primer R 0.4 µL cDNA 2 µL <![CDATA[ddH2O]]> 6.8 µL

[0170] Table 9 qRT-PCR reaction procedure

[0171]

[0172] 4) Western blot identification of transgenic plants

[0173] ①Total protein extraction

[0174] Total protein was extracted from tomato fruit using the Tris-saturated phenol method. 5 g of tomato peel powder, ground in liquid nitrogen, was weighed and added to 15 mL of protein extraction buffer (0.7 M Sucrose, 0.5 M Tris-HCl (pH=7.5), 0.1 M KCl, 0.5 mM EDTA, 1 mM PMSF, 1% PVPP, and 2% β-Mercaptoethanol). The mixture was vortexed for 15 min at low temperature to ensure thorough mixing. Then, 15 mL of Tris-saturated phenol was added, and the mixture was vortexed for 5 min to mix. The mixture was centrifuged at 20000×g for 15 min at 4℃. The supernatant phenolic phase was transferred to a new centrifuge tube, and an equal volume of protein extraction buffer was added. The tube was vortexed for 10 min. The mixture was centrifuged at 20000×g for 15 min at 4℃. The supernatant phenolic protein extract was then collected and 5 volumes of 0.1 M ammonium acetate methanol solution were added. The mixture was incubated overnight at -20℃. Centrifuge at 20000×g for 15 min at 4℃ and collect the protein precipitate. Then rinse twice with pre-cooled 0.1 M ammonium acetate methanol solution and acetone solution, respectively. Dry the protein precipitate in a clean bench and freeze for later use.

[0175] ②Western blot experiment

[0176] Total protein samples from tomato fruits were separated using a 10% SDS-PAGE gel and then transferred to a PVDF (Millipore) membrane using a semi-dry transfer device. The PVDF membrane was blocked for 1 h at room temperature with TBST buffer (blocking buffer) containing 5% (w / v) skim milk. It was then incubated for 1 h with blocking buffers containing anti-HA (Abmart) and anti-Actin (Abmart) antibodies, respectively. After antibody incubation, the membrane was washed four times with TBST buffer, 5 min each time. The washed PVDF membrane was then incubated for 1 h at room temperature with blocking buffer containing anti-mouse IgG secondary antibody (containing horseradish peroxidase HRP). Finally, the PVDF membrane was washed four more times with TBST buffer, and the HRP substrate (Pierce Biotechnology) was used to react with the antibodies bound to the PVDF membrane. Protein signals were detected and photographed using a chemiluminescence system (Tanon, 5200Multi).

[0177] 1.4 Identification of tolerance to high temperature stress in tomato fruits overexpressing the SlKUF7 gene and determination of corresponding indicators

[0178] 1.4.1 Observation of heat resistance phenotypes in tomato fruits

[0179] Five fruits from wild-type and SlKUF7 gene-overexpressing tomato lines (OE-14 and OE-19) were selected 45 days after flowering and treated in a 38℃ incubator for 3 days. After removal, the phenotypic changes of the fruits were observed, and representative fruit phenotypes were photographed and recorded.

[0180] 1.4.2 Tomato Fruit Firmness Measurement

[0181] Fruit firmness was measured using a pointer-type fruit firmness tester (Guangzhou Hangxin, GY-1). Four symmetrical cross-shaped measurement points were selected on the surface of the largest cross-section of the tomato fruit, and the firmness values ​​were measured at each point. The average of the four measurements was then used as the evaluation index for fruit firmness. Fruit firmness measurements were performed in triplicate, and the data were presented in a bar chart using the mean and standard deviation of the three measurements. Significance analysis between SlKUF7-overexpressing tomato fruits and wild-type fruits was conducted using a two-tailed Student's t-test (* represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001).

[0182] 1.4.3 Determination of abscisic acid (ABA) content in tomato fruits

[0183] Tomato fruit tissue was immediately frozen and ground into powder in liquid nitrogen. The fruit sample (50 mg) was dissolved in 1 mL of methanol / water / formic acid (15:4:1, v / v / v). An internal standard mixture was added to the mixture as a quantification internal standard. The mixture was vortexed for 10 min and centrifuged at 12000 g at 4 °C for 5 min. The supernatant was then collected and evaporated to dryness. The precipitate was dissolved in 80% (v / v) methanol and filtered for LC-MS / MS analysis.

[0184] ABA extract was extracted using a UHPLC-ESI-MS / MS system (ExionLC™ AD) equipped with Waters ACQUITY UHPLC HSS T3 C 18 The chromatographic column (100 mm × 2.1 mm, 1.8 µm) was used at a flow rate of 0.35 mL / min. -1 The temperature was 40℃. Mobile phase A was 0.04% aqueous acetic acid solution, and mobile phase B was 0.04% acetic acid-acetonitrile. ABA extract was separated using a linear gradient of 5% mobile phase B (1 min), 95% mobile phase B (8 min), and 5% mobile phase B (3 min).

[0185] ABA samples were then analyzed using a triple quadrupole linear ion trap mass spectrometer (QTRAP6500+) equipped with an ESI Turbo ion spray interface, operating in both positive and negative ion modes and controlled by Analyst 1.6.3 software (Sciex). ESI source operating parameters were: ion source, ESI+ / −; source temperature, 550 °C; ion spray voltage, 5500 V (positive), -4500 V (negative); and veil gas, 35 psi. ABA analysis was performed using a predetermined multiple reaction monitoring (MRM). Data acquisition and ABA quantification were performed using Analyst 1.6.3 software (Sciex) and Multiquant 3.0.3 software (Sciex), respectively. Further DP and CE optimizations were performed on the clustering potential (DP) and collision energy (CE) of individual MRM transitions. A specific set of MRM transitions was monitored for each epoch based on the metabolites eluted during this period. ABA identification and quantification were determined by retention times and dose-response profiles of standard samples. The standard curve equation for ABA content calculation is y = 0.09466x - 2.33275e -4 The correlation coefficient was 0.99767.

[0186] The ABA content determination was performed in three biological replicates, and the data were presented by plotting a bar chart using the mean and standard deviation of the three data sets. The significance analysis between SlKUF7 overexpressing tomato fruits and wild-type was performed using a two-tailed Student's t-test (* represents p-value < 0.05; ** represents p-value < 0.01; *** represents p-value < 0.001).

[0187] 2. Results and Analysis

[0188] 2.1 Identification of SlKUF7 gene overexpression in tomato fruits

[0189] To construct tomato plants overexpressing the SlKUF7 gene, this invention uses the pCambia1300 expression vector to construct a SlKUF7-HA fusion protein expression cassette driven by the constitutive promoter CaMV35S. For example... Figure 1 As shown in Figure A. After transformation of tomatoes with the overexpression vector, two tomato lines (OE-14 and OE-19) with overexpression of the SlKUF7 gene were obtained in the T2 generation.

[0190] like Figure 1 As shown in Figure B, qRT-PCR analysis revealed that the transcriptional level of SlKUF7 in OE-14 and OE-19 fruits was significantly higher than that in the wild type.

[0191] like Figure 1As shown in Figure C, immunoblotting confirmed that the SlKUF7-HA fusion protein was successfully expressed in OE-14 and OE-19 fruits, indicating that these two SlKUF7 gene overexpressing tomato lines can be used for subsequent gene function studies.

[0192] 2.2 Tolerance analysis of tomato fruits overexpressing the SlKUF7 gene under high temperature conditions

[0193] To determine the effect of the SlKUF7 gene on the heat tolerance of tomato fruits, this invention subjected wild-type and SlKUF7 gene-overexpressing red-ripe fruits to a high-temperature treatment of 38°C. Figure 2 As shown in Figure A, the results indicate that after high-temperature treatment, the peel of wild-type fruits cracked severely, while the fruits overexpressing the SlKUF7 gene (OE-14 and OE-19) maintained an intact appearance. Since peel firmness is directly related to fruit heat resistance, this invention analyzed the firmness of fruits overexpressing the SlKUF7 gene. Figure 2 As shown in Figure B, the results indicate that the firmness of both OE-14 and OE-19 fruits was significantly higher than that of wild-type fruits.

[0194] 2.3 Analysis of abscisic acid (ABA) content in tomato fruits overexpressing the SlKUF7 gene

[0195] This invention determined the endogenous ABA content in tomato fruits. For example... Figure 3 As shown, the results indicate that, compared to the wild type, the ABA levels in OE-14 and OE-19 fruits were significantly increased.

[0196] These results collectively suggest that the SlKUF7 gene may enhance the heat resistance of tomato fruits by increasing the endogenous ABA content.

[0197] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An application of a tomato SlKUF7 protein in any of the following: (1) Application in regulating the heat resistance of tomato fruits; (2) Application in the cultivation of heat-resistant transgenic tomatoes; (3) Application in the preparation of products that improve the heat resistance of tomato fruits; The amino acid sequence of the tomato SlKUF7 protein is shown in SEQ ID NO.

2.

2. The application as described in claim 1, characterized in that, Upregulating the expression level of the tomato SlKUF7 protein in tomatoes improves the heat resistance of tomato fruits.

3. The use of the gene encoding the tomato SlKUF7 protein as described in claim 1 in any of the following: (1) Application in regulating the heat resistance of tomato fruits; (2) Application in the cultivation of heat-resistant transgenic tomatoes; (3) Application in the preparation of products that improve the heat resistance of tomato fruits; The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

4. The application as described in claim 3, characterized in that, Upregulating the expression of the encoded gene in tomatoes improves the heat resistance of tomato fruits.

5. An application of a gene overexpression vector, characterized in that, The overexpression vector contains the gene encoding the tomato SlKUF7 protein; The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1; The application is any one of the following: (1) Application in regulating the heat resistance of tomato fruits; (2) Application in the cultivation of heat-resistant transgenic tomatoes; (3) Application in the preparation of products that improve the heat resistance of tomato fruits.

6. The use of an engineered bacterium comprising the overexpression vector of claim 5 in any of the following: (1) Application in regulating the heat resistance of tomato fruits; (2) Application in the cultivation of heat-resistant transgenic tomatoes; (3) Application in the preparation of products that improve the heat resistance of tomato fruits.

7. The application as described in any one of claims 1-6, characterized in that, The indicators of fruit heat resistance include fruit firmness.

8. A method for improving the heat resistance of tomato fruit, characterized in that, The method includes the step of upregulating the expression level of the gene encoding the tomato SlKUF7 protein in tomatoes to improve the heat resistance of the tomato fruit; The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

9. A breeding method for a heat-resistant transgenic tomato, characterized in that, Includes the following steps: The gene encoding the tomato SlKUF7 protein was overexpressed in tomato cells, the tomato cells were then cultured, and tomatoes were regenerated using the tomato cells to obtain heat-resistant transgenic tomatoes. The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

10. The method as described in claim 8 or 9, characterized in that, The indicators of fruit heat resistance include fruit firmness.

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