Application of SIPTP1b gene in regulating postharvest cold resistance of tomato fruit

Abstract

This invention discloses SIPTP1b The application of genes in regulating the postharvest cold resistance of tomato fruits falls under the field of genetic engineering technology. SIPTP1b The CDS nucleotide sequence of the gene is shown in SEQ ID NO.1; by inhibiting SIPTP1b Gene expression enhances the postharvest cold resistance of tomato fruits through overexpression. SIPTP1b Genes that reduce the postharvest cold resistance of tomato fruits. This invention discloses for the first time... SIPTP1b Genes can negatively regulate AsA synthesis in tomato fruits and postharvest cold resistance, and can be inhibited by... SIPTP1b Improving the postharvest cold resistance of tomato fruits through gene expression provides a new approach to fruit and vegetable preservation technology and a new path for breeding tomatoes with postharvest cold resistance. It has significant industrial application value and solves the problem that existing tomato fruits are easily affected by low temperature stress during postharvest storage, which seriously affects fruit quality and shelf life.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to SIPTP1b Application of genes in regulating the postharvest cold resistance of tomato fruits. Background Technology

[0002] tomato( Solanum lycopersicum Ascorbyl is an important global economic crop. Its fruits are susceptible to low-temperature stress during post-harvest storage, leading to chilling injury, manifested as impaired color change, accumulation of reactive oxygen species (ROS), and membrane lipid peroxidation, which seriously affect fruit quality and shelf life. Ascorbic acid (AsA), as an important antioxidant in plants, plays a crucial role in scavenging ROS and maintaining redox balance.

[0003] Existing research indicates that protein tyrosine phosphatases (PTPs) participate in plant stress responses by regulating the MAPK signaling pathway through dephosphorylation modification. For example, Arabidopsis thaliana AtPTP1 and potato StPTP1a have been shown to participate in stress responses. However, the function of PTPs in postharvest low-temperature stress in tomato fruit and the molecular mechanism by which they regulate AsA synthesis remain unclear. SIPTP1b The role of genes in regulating the postharvest cold resistance of tomato fruits has been rarely reported. Summary of the Invention

[0004] To solve the above-mentioned technical problems, the purpose of this invention is to provide... SIPTP1b The application of genes in regulating the postharvest cold resistance of tomato fruits aims to address the problems of low-temperature stress during postharvest storage of tomato fruits, which seriously affects fruit quality and shelf life.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A first aspect of the present invention provides SIPTP1b Application of genes in regulating postharvest cold resistance of tomato fruits SIPTP1b The CDS nucleotide sequence of the gene is shown in SEQ ID NO.1: ATGGCCACCGCCGGTAAACTCGTGTCGTCGTCGTCGTCGTCACCCAAGCCTTTCGATTTCTCCGACGATTCAGTTCCTAAGAGAGTCGTTCTGTCGAGTGATCAGCAGCGTTATTGCTTGGAAGTACTCAAAGTCTTCAAGGACAAGAGGTTTTCTGCTCCCGAAAAAATCCGCCAAGAGTTCATGACGTTGCAGGCAACTAGGATGAGAGCTTCAGAAATGAAAAGTAGATGCTCAATGGCTTTGAACAGCGCAAACATTAGCAAAAATCGATACACTGATGTTCTGCCATTTGACAATAACAGGGTTGTTTTGGACCCACCAGCTAGAGGATATATAAATGCAAGCTTCATTAAGATATCTGAAGACGTGTCTCAGTTTATTGCAACACAAGGTCCTCTACAACACACTTTTGAAGACTTCTGGGAAATGATAATCCAGCATCGCTGTCCTGTGATTGTGATGCTTACACAATTGTTTGACAACTACAAGATTGTCAAGTGTGGGGATTATTTTCAGGCAGATGGTGGTCCCAGAAGATTTGGCAATATATGTATTGTTACGAAGTGGATAAAGACGACTCAGACTTCATTGATTTTGCGATGTCTGGAGGTGAACTATATTGAGTCAACAGAACCACCTTTGTGTGTTTTACACATTCAATATCCCGACTGGCCTGACCATGGAGTTCCAAAGGACACTCTTGCTGTTCGTGAAATACTGAAACAAACATTTAGTGTCCCACCCAGTCTTGGACCAATTGTGGTGCACTGCAGTGCAGGTATTGGTAGGACTGGAACATACTGTGCAATCCATAATACCATCCAAAGAATATTGGGTGGAGATAAGTCTGCTCTAGATCTAGTTAACACAATAACCATTTTTAGGTCTCAGCGAATTGGGATGGTTCAAACCATGGAGCAATACCTCTTTTGCTATGATGCCGTTATCGATGAGCTTGAAGACCTCATTTCAGATAGCCAATGA(SEQ ID NO.1)。

[0006] Furthermore, by inhibiting SIPTP1b Gene expression enhances the postharvest cold resistance of tomato fruits through overexpression. SIPTP1b Genes that reduce the postharvest cold resistance of tomato fruits.

[0007] Furthermore, SIPTP1b The protein sequence encoded by the CDS nucleotide sequence of the gene is shown in SEQ ID NO.2: MATAGKLVSSSSSSPKPFDFSDDSVPKRVVLSSDQQRYCLEVLKVFKDKRFSAPEKIRQEFMTLQATRMRASEMKSRCSMALNSANISKNRYTDVLPFDNNRVVLDPPARGYINASFIKISEDVSQFIATQGPLQHTFEDFWEMIIQHRCPVIVMLTQLFDNYKIV KCGDYFQADGGPRRFGNICIVTKWIKTTQTSLILRCLEVNYIESTEPPLCVLHIQYPDWPDHGVPKDTLAVREILKQTFSVPPSLGPIVVHCSAGIGRTGTYCAIHNTIQRILGGDKSALDLVNTITIFRSQRIGMVQTMEQYLFCYDAVIDELEDLISDSQ (SEQ ID NO.2).

[0008] A second aspect of the present invention provides a method for improving the postharvest cold resistance of tomato fruits, comprising the following steps: Build SIPTP1b Gene repression expression vectors were used to transform tomatoes, and transgenic plants were constructed. SIPTP1b The CDS nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0009] Furthermore, the expression suppression vectors include RNAi interference vectors or CRISPR / Cas9 knockout vectors.

[0010] A third aspect of the invention provides SIPTP1b Application of gene repressor expression vectors in improving postharvest cold resistance of tomato fruits; the repressor expression vector is a gene repressor expression vector. SIPTP1b Designed with genes as targets SIPTP1b Gene RNAi interference vectors or CRISPR / Cas9 knockout vectors SIPTP1b The CDS nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0011] Furthermore, SIPTP1bThe gene RNAi interference vector was constructed using the following method: first targeting... SIPTP1b A specific interference fragment was designed based on the CDS nucleotide sequence of the gene, then recombined into an intermediate vector via a backpropagation (BP) reaction, and finally transferred into an RNA interference expression vector via a backpropagation (LR) reaction to obtain the desired result. SIPTP1b Gene RNAi interference vector.

[0012] Furthermore, SIPTP1b The gene CRISPR / Cas9 knockout vector was constructed using the following method: first, a targeted gene was designed. SIPTP1b The sgRNA sequence of the gene exons was obtained, and then the sgRNA expression cassette and Cas9 expression cassette were assembled into a vector using an enzyme digestion and ligation system to obtain... SIPTP1b Gene CRISPR / Cas9 knockout vector.

[0013] The present invention has the following beneficial effects: 1. This invention discloses for the first time SIPTP1b Genes can negatively regulate AsA synthesis and postharvest cold resistance in tomato fruits, providing a genetic basis for regulating postharvest cold resistance in tomato fruits.

[0014] 2. This invention is based on SIPTP1b Gene editing or RNAi strategies targeting genes were constructed. SIPTP1b The gene interference expression vector was developed, and a method for improving the postharvest cold resistance of tomato fruits based on the vector was provided. This method can be applied to improve the postharvest cold resistance of tomatoes and other fruits and vegetables.

[0015] 3. This invention inhibits SIPTP1b Gene expression can improve the postharvest cold resistance of tomato fruits, providing new ideas for fruit and vegetable preservation technology and a new path for breeding tomatoes with postharvest cold resistance, which has important industrial application value. Attached Figure Description

[0016] Figure 1 for SIPTP1b A graph showing the results of bioinformatics analysis of genes; Figure 2 for SIPTP1b The results of gene expression pattern analysis are shown in the figure. In the figure, A represents the relative expression level in different tissues, and B represents the expression level in different developmental stages of the fruit. Figure 3 for SIPTP1b Gene overexpression and RNA interference in strains SIPTP1b A schematic diagram of gene expression results and the editing method of knockout lines, where A represents the relative expression level and B represents the CRISPR / Cas9 knockout vector. Figure 4 Low temperature treatment experiment SIPTP1b The graph shows the transcriptional and protein levels of genes, where A represents the transcriptional level and B represents the protein level. Figure 5 Phenotypic images of fruits from different strains after low-temperature stress treatment; Figure 6 The graph shows the malondialdehyde (MDA) content and peroxidase activity of fruits from different strains after low-temperature stress treatment. In the graph, A represents MDA content and B represents peroxidase activity. Figure 7 The graph shows the results of ascorbic acid (AsA) activity determination in fruits of different strains after low-temperature stress treatment. In Figure A, low-temperature stress rapidly induced AsA accumulation in WT-damaged fruits; Figure B shows the results of low-temperature stress treatment. SlPTP1b Different strains of fruit were subjected to inhibition of genes related to ascorbic acid synthesis. SIMYB11 , SIGalDH and SIDHAR The relative expression levels are shown in Figure C, which is a comparison of the reduced ascorbic acid content of fruits from different strains under low temperature stress and control treatment, and Figure D is a comparison of the total ascorbic acid content of fruits from different strains under low temperature stress and control treatment. Figure 8 The image shows the results of the detection of reactive oxygen species in tomato peel. Figure 9 The figures show the results of yeast two-hybrid assay, luciferase complementation assay, and immunoprecipitation assay. In the figure, A is the result of yeast two-hybrid assay, B is the result of luciferase complementation assay, and C is the result of immunoprecipitation assay. Figure 10 The figures show the results of the phosphatase activity assay. In the figure, A is a schematic diagram of the vector construction, B is a graph of the GST affinity chromatography purification results, C is a graph of phosphatase activity assay, D is a graph of absorbance change at 405 nm wavelength, and E is a graph of absorbance change at 405 nm wavelength under H2O2 inhibition conditions. Figure 11 The first image shows the results of the in vivo dephosphorylation experiment, while the second image shows the results of the Western blotting analysis. Figure 12 The diagram shows the experimental results of the dual-luciferase reporter gene assay. In the diagram, A is a schematic diagram of the vector, and B is the detection result of the dual-luciferase reporter gene assay. Detailed Implementation

[0017] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0018] Example 1: Tomato SIPTP1bGene cloning and expression analysis (1) Gene cloning Tomato ( Solanum lycopersicum Total RNA was extracted from the fruit of the Micro-Tom variety during its color-breaking stage. RNA was extracted using the Eastep® Super Total RNA Extraction Kit, and the first strand of cDNA was synthesized using the HiScript II Q RTSuperMix reverse transcription kit. Using this cDNA as a template, PCR amplification was performed using PrimeSTAR® Max DNA Polymerase high-fidelity enzyme. The amplified RNA was then analyzed from the tomato genome database (https: / / solgenomics.net). SIPTP1b Design specific primers based on the coding sequence (CDS) of the gene (gene number: Solyc06g065480): Forward primer: 5'-ATGCCACCGCCGGTAAAC-3' (SEQ ID NO.3); Reverse primer: 5'-TCATTGGCTATCTGAAATGAGG-3' (SEQ ID NO.4).

[0019] The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 30 s, for a total of 30 cycles; and a final extension at 72℃ for 5 min.

[0020] A PCR product of 987 bp was amplified, recovered by agarose gel electrophoresis, ligated into the pEASY-Blunt Zero cloning vector, transformed into E. coli DH5α, and positive clones were screened for sequencing verification to obtain the product containing... SIPTP1b Recombinant plasmid of the CDS gene. Sequence analysis results are as follows: Figure 1 As shown, the results indicate that the gene encodes a protein containing 328 amino acids with a molecular weight of 37.12 kDa and an isoelectric point of 7.01. It possesses a typical protein tyrosine phosphatase (PTP) catalytic domain (PTPc) and a conserved motif (H / V)C(X)5R(S / T).

[0021] (2) Analysis of expression patterns To clarify SIPTP1b To determine the tissue-specific expression of the gene, RNA was extracted from the roots, stems, leaves, flowers, and fruits at different developmental stages (immature green fruit, mature green fruit, color-breaking stage, and red fruit) of wild-type tomatoes. The expression levels were detected by real-time quantitative PCR (RT-qPCR). The tomato Actin gene (SIACTIN) was used as an internal control, and the primer sequences were as follows: qSIPTP1b-F: 5'-GCCTTTTCCGATTTCTCCGACG-3' (SEQ ID NO. 5); qSIPTP1b-R: 5'-GCTCTCATCCTAGTTGCCTGC-3' (SEQ ID NO. 6).

[0022] The results are as follows Figure 2 As shown, SIPTP1b The gene is expressed in all tissues of tomatoes, but its expression level is highest in fruits during the color-breaking and red-fruiting stages, indicating that the gene may play an important role in fruit ripening.

[0023] Example 2: SIPTP1b Functional verification of the gene in regulating cold tolerance and ascorbic acid synthesis in tomato fruit (1) Construction of transgenic tomato lines 1) Construction of overexpression vectors and genetic transformation Using homologous recombination, the cloned in Example 1 SIPTP1b CDS was inserted between the Sma I and Sal I restriction sites of the plant overexpression vector pLP100-35S to construct the 35S::SIPTP1b-HA recombinant plasmid. This plasmid was transformed into Agrobacterium GV3101, and then into tomato (Micro-Tom) cotyledonary explants via Agrobacterium-mediated genetic transformation. The transformed explants were induced to develop resistant adventitious shoots on 2Z selection medium containing 50 mg / L kanamycin. After subculturing, the elongated adventitious shoots were cut off and transferred to ENR rooting medium to induce rooting. RT-qPCR was used to detect the presence of [unclear - likely a specific enzyme or component] in the leaves. SIPTP1b The expression levels were determined, and T1 generation overexpression lines with significantly elevated expression levels were selected and named OE-SIPTP1b#1 and OE-SIPTP1b#2. The relative expression levels of the overexpression lines are as follows: Figure 3 As shown in Figure A.

[0024] 2) Construction and genetic transformation of RNA interference vectors against SIPTP1b A specific interference fragment was designed using gene CDS, recombined into the pDONR207 vector via a backpropagation (BP) reaction, and then transferred to the RNA interference expression vector pJE042 via a backpropagation (LR) reaction. Tomatoes were transformed using the above Agrobacterium-mediated transformation method, and T1 generation transgenic plants were obtained through Kana selection. RT-qPCR was used to screen for... SIPTP1b The RNA interference lines with significantly reduced expression levels were named RNAi-SIPTP1b#3 and RNAi-SIPTP1b#12. The relative expression levels of the RNA interference lines are as follows: Figure 3 As shown in Figure A.

[0025] 3) Construction of CRISPR / Cas9 gene knockout mutants Design two targets SIPTP1b The sgRNA sequences of the gene exons are shown in SEQ ID NO.7-8, respectively: sgRNA-1: 5'-ATATCAGCGATGGCCACCG-3' (PAM: CGG), sgRNA-2: 5'-TCAAGTCTTCAAGGACAAG-3' (PAM: AGG).

[0026] The sgRNA expression cassette and Cas9 expression cassette were assembled into the pDGB3 vector using the Golden Braid restriction enzyme ligation system. After transformation into tomatoes, the target region of the T1 generation plants was amplified by PCR and identified by sequencing. A homozygous mutant, slptp1b#2, was obtained. This mutant has a 13 bp deletion at target site 1, resulting in the loss of the start codon and failure to initiate protein translation. Figure 3 As shown in Figure B.

[0027] (2) Postharvest low temperature stress treatment and phenotypic analysis of tomato fruits Wild-type (WT), overexpressing (OE-SIPTP1b#1, #2), RNA interference (RNAi-SIPTP1b#3, #12), and knockout mutant (slptp1b#2) tomato fruits were selected at the color-breaking stage (fruit surface begins to turn red). The fruits were placed in a 4℃ incubator for 14 days of low-temperature treatment, followed by recovery at 25℃ for 14 days. Fruits stored at 25℃ for 28 days served as the control group. At least nine fruits were treated for each line, with three replicates.

[0028] The results of the low-temperature treatment experiment showed SIPTP1b Transcriptional and protein levels were significantly downregulated within 1 hour, indicating a response to low-temperature stress. Figure 4 ).

[0029] Observe the fruit's appearance and phenotype; the results are as follows: Figure 5 As shown, after low-temperature treatment, WT fruits showed slight color change impairment; OE-SIPTP1b fruits showed severe chilling injury symptoms, and the fruits could not turn red normally, appearing greenish-yellow; while RNAi-SIPTP1b and slptp1b fruits showed normal color change, with no significant difference from the room temperature control group, showing stronger cold resistance.

[0030] (3) Measurement of physiological indicators 1) Determination of malondialdehyde (MDA) content Take 0.2 g of tomato peel after low-temperature treatment, add 2 mL of 10 wt% trichloroacetic acid (TCA), grind into a homogenate, centrifuge, and collect the supernatant. Add an equal volume of 0.6 wt% thiobarbituric acid (TBA), react in a boiling water bath for 15 min, and measure the OD after cooling. 532 OD 600 and OD 450 Value, calculate MDA content.

[0031] The results are as follows Figure 6 As shown in Figure A, the MDA content of OE-SIPTP1b fruit was significantly higher than that of WT, while the MDA content of RNAi-SIPTP1b and slptp1b fruit was significantly lower than that of WT.

[0032] 2) Peroxidase (POD) activity assay Crude enzyme solution was extracted from tomato peel, and the reaction was initiated by adding 2 wt% H2O2 using guaiacol as a substrate. The OD was then measured. 470 The rate of change of the value within 1 minute is used to calculate the POD activity.

[0033] The results are as follows Figure 6 As shown in Figure B, the POD activity of OE-SIPTP1b fruit was significantly lower than that of WT, while the POD activity of RNAi-SIPTP1b and slptp1b fruit was significantly higher than that of WT.

[0034] 3) Ascorbic acid (AsA) activity assay Take 0.2 g of tomato peel powder and add 1 mL of 6 wt% TCA to extract AsA. Use Fe... 3+ The total AsA and reduced AsA content was determined by the bipyridine colorimetric method.

[0035] The results are as follows Figure 7 As shown, after treatment at 4℃ for 24 h, compared with WT, the total AsA and reduced AsA content in OE-SIPTP1b fruits was significantly reduced, while the AsA content in RNAi-SIPTP1b and slptp1b fruits was significantly increased. Simultaneously, RT-qPCR detection indicated that the key enzyme gene for AsA synthesis... SIGalDH and SIDHAR The expression level of AsA showed a consistent trend with the change in AsA content.

[0036] 4) Determination of reactive oxygen species (ROS) content The ROS content in tomato peel was determined using the DCFH-DA fluorescent probe method.

[0037] The results are as follows Figure 8As shown, after low-temperature stress, the ROS fluorescence intensity in RNAi-SIPTP1b and slptp1b fruits was significantly lower than that in WT fruits, while the ROS fluorescence intensity in OE-SIPTP1b fruits was significantly higher than that in WT fruits. This indicates that... SIPTP1b Negative regulation of AsA's ability to scavenge ROS.

[0038] Example 3: SIPTP1b and SIMAPK8 Protein-protein interactions and functional verification (1) Yeast two-hybrid (Y2H) verification of protein interactions Will SIPTP1b The CDS was cloned into the pGADT7 vector (AD-SIPTP1b), and SIMAPK8 The CDS was cloned into the pGBKT7 vector (BD-SIMAPK8). It was co-transformed into yeast strain Y2HGold, and positive clones were screened on SD / -Trp / -Leu (DDO) medium. Positive yeast colonies were picked, spotted onto SD / -Trp / -Leu / -His / -Ade (QDO) quadruple-deficient medium, and detected by X-α-gal staining.

[0039] The results showed that yeast cells co-transformed with AD-SIPTP1b and BD-SIMAPK8 could grow normally on QDO medium and showed a blue color ( ). Figure 9 (Figure A in the middle) indicates SIPTP1b and SIMAPK8 There are interactions within yeast.

[0040] (2) Validation by luciferase complementation assay (LCA) Will SIPTP1b The CDS is integrated with nLUC (JW771). SIMAPK8 CDS was fused with cLUC (JW772) and transformed into Agrobacterium GV3101. Equal volumes of Agrobacterium cultures containing different recombinant plasmids were mixed and injected into leaves of *Nicotiana benthamiana*. Co-expression of SIMAPK8-nLUC and SLARF4-cLUC was used as positive controls. Three days post-injection, a 1 mmol / L fluorophore substrate was applied to the underside of the leaves. After a 2-minute reaction in the dark, fluorescence signals were detected using a plant in vivo imaging system.

[0041] The results showed that strong fluorescence signals were detected in tobacco leaf regions co-expressing SIPTP1b-nLUC and SIMAPK8-cLUC. Figure 9 Figure B in the middle section confirms that the two have a specific interaction within the plant.

[0042] (3) Verification of co-immunoprecipitation (Co-IP) The constructed plant expression vectors 35S::SIPTP1b-GFP and 35S::SIMAPK8-Flag were co-transformed into Nicotiana benthamiana. Total protein was extracted, and immunoprecipitation was performed using Anti-GFP magnetic beads. The precipitated complex was separated by SDS-PAGE and detected by Western blotting using Anti-Flag antibody.

[0043] The results showed that a specific band of SIMAPK8-Flag could be detected in the SIPTP1b-GFP immunoprecipitation complex, while no band was detected in the GFP empty control group. Figure 9 (Figure C in the middle) further proves SIPTP1b and SIMAPK8 Interactions within the plant.

[0044] (4) Phosphatase activity detection Will SIPTP1b CDS and its active site mutant (D227A / C258S, named SIPTP1bAS) were cloned into the pGEX-4T-1 vector ( Figure 10 (Figure A) was transformed into *E. coli* TSsetta (DE3) and induced with 0.2 mmol / L IPTG at 16℃ for 20 h. The GST-SIPTP1b and GST-SIPTP1bAS fusion proteins were purified by GST affinity chromatography. Figure 10 (Figure B in the middle). The absorbance change was detected at a wavelength of 405 nm using p-nitrophenyl phosphate (pNPP) as a substrate.

[0045] The results showed that GST-SIPTP1b protein can efficiently hydrolyze pNPP to produce a yellow product ( Figure 10 (Figure C) and the absorbance increased linearly with time; while neither GST-SIPTP1bAS protein nor GST empty vector showed this activity. Figure 10 (See Figure D). Simultaneously, the addition of 200 mmol / L H2O2 rapidly inhibited the phosphatase activity of GST-SIPTP1b (…). Figure 10 (Figure E in the image). This indicates that SIPTP1b protein is a redox-regulated functional protein tyrosine phosphatase.

[0046] (5) In vivo dephosphorylation experiment SIMAPK8-Flag and SIPTP1b-GFP or empty GFP vectors were co-expressed in *Nicotiana benthamiana*. Total protein was extracted, immunoprecipitated with anti-Flag magnetic beads, and detected by Western blotting using a pERK antibody that specifically recognizes phosphorylated tyrosine (recognizing pTEpYmotif).

[0047] The results are as follows Figure 11As shown, when SIMAPK8 protein kinase and SIPTP1b protein tyrosine phosphatase are co-expressed, the phosphorylation signal intensity is significantly lower than that of the control group co-expressed with GFP empty vector. This indicates that SIPTP1b protein tyrosine phosphatase can perform tyrosine dephosphorylation modification of SIMAPK8 protein kinase in vivo.

[0048] (6) Dual-Luciferase Reporter Gene Assay (Dual-LUC) The effector vectors (35S::SIMYB11, 35S::SIMAPK8, 35S::SIPTP1b, or 35S::SIPTP1bAS) and reporter vectors (SIGalDH or SIDHAR promoters driving the firefly luciferase gene, with the 35S-driven Renilla luciferase gene as an internal control) were mixed in a certain ratio and transformed into Agrobacterium tumefaciens, which was then injected into leaves of Nicotiana benthamiana. Three days after injection, leaf discs were lysed, and fluorescence values ​​(Firefly / Renilla) were measured on a microplate luminescence analyzer using a dual-luciferase reporter gene assay kit.

[0049] The results are as follows Figure 12 As shown, expressed separately SIMYB11 It can mildly activate reporter genes; co-expression SIMAPK8 and SIMYB11 It can significantly enhance the transcriptional activation activity of reporter genes; and when expressed simultaneously... SIPTP1b , SIMAPK8 and SIMYB11 hour, SIMAPK8 right SIMYB11 The enhancing effect was significantly inhibited; however, the co-expression of inactivated SIPTP1bAS did not show this inhibitory effect. This indicates that SIPTP1b protein tyrosine phosphatase inhibits the kinase activity of SIMAPK8 protein kinase through dephosphorylation, thereby negatively regulating the transcriptional activation of AsA synthesis genes by the SIMAPK8-SIMYB11 module.

[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. SIPTP1b The application of genes in regulating the postharvest cold resistance of tomato fruits is characterized by, By inhibiting SIPTP1b Gene expression enhances the postharvest cold resistance of tomato fruits, or through overexpression. SIPTP1b Genes that reduce the postharvest cold resistance of tomato fruits; SIPTP1b The CDS nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A method for improving the postharvest cold resistance of tomato fruits, characterized in that, Includes the following steps: Build SIPTP1b Gene repression expression vectors were used to transform tomatoes, and transgenic plants were constructed. The SIPTP1b The CDS nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. The method according to claim 2, characterized in that, The suppression expression vector includes an RNAi interference vector or a CRISPR / Cas9 knockout vector.

4. SIPTP1b The application of gene repression expression vectors in improving the postharvest cold resistance of tomato fruits is characterized by, The SIPTP1b Gene repression vectors are based on SIPTP1b Designed with genes as targets SIPTP1b Gene RNAi interference vector or CRISPR / Cas9 knockout vector, the SIPTP1b The CDS nucleotide sequence of the gene is shown in SEQ ID NO.

1.

5. The application according to claim 4, characterized in that, The SIPTP1b The gene RNAi interference vector was constructed using the following method: first targeting... SIPTP1b A specific interference fragment was designed based on the CDS nucleotide sequence of the gene, then recombined into an intermediate vector via a backpropagation (BP) reaction, and finally transferred into an RNA interference expression vector via a backpropagation (LR) reaction to obtain the desired result. SIPTP1b Gene RNAi interference vector.

6. The application according to claim 4, characterized in that, The SIPTP1b The gene CRISPR / Cas9 knockout vector was constructed using the following method: first, a targeted gene was designed. SIPTP1b The sgRNA sequence of the gene exon was obtained, and then the sgRNA expression cassette and Cas9 expression cassette were assembled into the vector using an enzyme digestion and ligation system to obtain... SIPTP1b Gene CRISPR / Cas9 knockout vector.

Images