Application of SlLNC402-SlJAZ7 / SlMYC2-SlREF model in regulating tomato pollen response to high temperature stress

Abstract

The invention provides an application of an SlLNC402-SlJAZ7 / SlMYC2-SlREF model in regulating tomato pollen to respond to high temperature stress, the SlLNC402-SlJAZ7 / SlMYC2-SlREF thermal response model is predicted and identified by using complete transcriptome data of tomato flower buds under high temperature stress, and the SlLNC402-SlJAZ7 / SlMYC2-SlREF thermal response model is obtained by cloning a full-length sequence of a coding region of SlLNC402 and a coding sequence of an SlREF gene. Yeast single hybrid and dual luciferase tests are adopted to verify the interaction regulation and control relationship between SlLNC402-SlJAZ7 and SlMYC2-SlREF, specifically including cloning of SlLNC402, cloning of SlMYC2, interaction verification of SlLNC402-SlJAZ7, interaction verification of SlMYC2-SlREF, and functional verification of SlREF in response to high temperature stress in tomato pollen, and prove that the long-chain non-coding SlLNC402 enhances the heat resistance of tomatoes by regulating and controlling a JA signal pathway, so that the tomato pollen resistance is enhanced, and the tomato pollen resistance is enhanced. The regulation and control model participates in a functional mechanism of responding to high temperature stress in the tomato bud stage, so that tomato germplasm resources can break through restrictions of factors such as regions and seasons on tomato yield and quality, and the purpose of annual balanced supply of tomatoes is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of a SlLNC402-SlJAZ7 / SlMYC2-SlREF model in regulating tomato pollen response to high temperature stress. Background Art

[0002] Tomato seed industry is an important factor in the tomato industry. However, compared with the advanced countries in the world, my country's tomato seed industry still has many gaps. The main problem is the lack of original innovation ability and few original achievements. At present, almost all the disease-resistant genes and high-quality genes used in domestic tomato breeding come from the separation of foreign varieties. Although the country has increased the systematic collection and identification of germplasm resources during the "Thirteenth Five-Year Plan", it is still in its infancy and lacks in-depth and systematic research. In particular, global warming has led to the frequent occurrence of extreme weather such as high temperature and drought, further curbing the momentum of the development of high-tech tomato seed industry.

[0003] The impact of high temperature stress caused by global warming on vegetable production is becoming increasingly serious. High temperature and drought stress severely limit the yield of vegetables and bring huge losses to the agricultural economy. Therefore, high temperature has become one of the important limiting factors affecting the development of the tomato industry. High temperature stress during the bud stage of tomatoes will cause poor development of flower organs, and it is easy to have poor flowering and fruit setting, and drop flowers and fruits, resulting in low tomato yield and poor quality. In recent years, important progress has been made in the study of plant high temperature stress, mainly focusing on the molecular mechanisms of transcription factors such as the heat shock transcription factor family and their regulatory genes. However, research on the epigenetic regulatory mechanism of non-coding RNA involved in high temperature stress response is still scarce. In addition, most of the current research on high temperature stress focuses on short-term heat stress treatment, which is difficult to apply to crop breeding. Therefore, the use of long-term high temperature stress to study the bud stage of tomatoes has become one of the important scientific hotspots, which is of great significance to improving the fruit setting rate and quality of tomatoes. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention aims to provide an application of the SlLNC402-SlJAZ7 / SlMYC2-SlREF model in regulating the response of tomato pollen to high temperature stress, and predicts and identifies the SlLNC402-SlJAZ7 / SlMYC2-SlREF thermal response model by using the full transcriptome data of tomato flower buds under high temperature stress, and validates the interaction and regulation relationship between SlLNC402-SlJAZ7 and SlMYC2-SlREF by cloning the full-length sequence of the coding region of SlLNC402 and the coding sequence of the SlMYC2 gene using yeast single-hybrid and dual-luciferase assays. Specifically, it includes the cloning of SlLNC402, the cloning of SlMYC2, the verification of the interaction between SlLNC402-SlJAZ7 and SlMYC2-SlREF, and the functional verification of SlREF in the response of tomato pollen to high temperature stress.

[0005] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0006] Application of SlLNC402-SlJAZ7 / SlMYC2-SlREF model in regulating tomato pollen response to high temperature stress,

[0007] The SlLNC402-SlJAZ7 / SlMYC2-SlREF model includes the long non-coding RNA SlLNC402, the SlMYC2 gene, the promoter of the SlJAZ7 gene and the promoter of the SlREF gene;

[0008] The SlLNC402-SlJAZ7 / SlMYC2-SlREF model is positively regulated in tomato pollen response to high temperature stress;

[0009] The nucleotide sequence of S1LNC402 is shown in SEQ ID NO.1;

[0010] The nucleotide sequence of the S1MYC2 gene is shown in SEQ ID NO.2;

[0011] The promoter nucleotide sequence of the SlJAZ7 gene is shown in SEQ ID NO.3;

[0012] The promoter nucleotide sequence of the SIREF gene is shown in SEQ ID NO.4.

[0013] Preferably,

[0014] The SlLNC402 positively regulates the expression of the SlJAZ7 gene and negatively regulates the response of tomato pollen to high temperature stress;

[0015] The SlMYC2 positively regulates the expression of the SlREF gene and positively regulates the response of tomato pollen to high temperature stress.

[0016] Preferably,

[0017] The promoters of the SlLNC402 and SlJAZ7 genes interact positively; the promoters of the SlMYC2 and SlREF genes interact positively.

[0018] Preferably,

[0019] The primers for amplifying S1LNC402 are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0020] Preferably,

[0021] The primers for amplifying the SlMYC2 gene are shown in SEQ ID NO.7 and SEQ ID NO.8.

[0022] Preferably,

[0023] The primers for amplifying the promoter of the SlJAZ7 gene are shown in SEQ ID NO.9 and SEQ ID NO.10.

[0024] Preferably,

[0025] The primers for amplifying the promoter of the SIREF gene are shown in SEQ ID NO.11 and SEQ ID NO.12.

[0026] A method for improving the response of tomato pollen to high temperature stress,

[0027] By reducing the expression of SlLNC402, the expression of the SlJAZ7 promoter was reduced, thereby relieving the inhibition of the SlMYC2 gene, increasing the expression of SlREF, and ultimately improving the high temperature resistance of tomatoes;

[0028] The nucleotide sequence of S1LNC402 is shown in SEQ ID NO.1;

[0029] The nucleotide sequence of the SlMYC2 gene is shown in SEQ ID NO.2.

[0030] Preferably,

[0031] The primers for fluorescence quantification of S1LNC402 are shown in SEQ ID NO.13 and SEQ ID NO.14.

[0032] The primers for SlJAZ7 fluorescence quantification are shown in SEQ ID NO.15 and SEQ ID NO.16.

[0033] The primers for SlMYC2 fluorescence quantification are shown in SEQ ID NO.17 and SEQ ID NO.18.

[0034] The primers for SlREF fluorescence quantification are shown in SEQ ID NO.19 and SEQ ID NO.20.

[0035] The full-length sequence of the SlJAZ7 gene coding region is shown in SEQ ID NO.21.

[0036] The full-length sequence of the SlREF gene coding region is shown in SEQ ID NO.22.

[0037] The beneficial effects of the present invention are:

[0038] The present invention uses the combined analysis of multi-omics data such as tomato flower bud mRNA, miRNA, lncRNA, circRNA, etc. under long-term high temperature stress, and finds that the long non-coding SlLNC402 enhances the heat tolerance of tomatoes by regulating the JA metabolic pathway. A SlLNC402-SlJAZ7 / SlMYC2-SlREF regulatory model is established. By studying the regulatory mode of SlLNC402 on the JA metabolic pathway in the regulatory model, identifying the downstream target genes of the core gene SlMYC2, and verifying the functions of the corresponding transformed strains, the functional mechanism of the non-coding RNA and mRNA regulatory models involved in the response of tomato to high temperature stress at the bud stage is revealed, so that tomato germplasm resources can break through the constraints of regional and seasonal factors on tomato yield and quality, and achieve the goal of year-round balanced supply of tomatoes. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0040] Figure 1 This is the effect of high temperature on the endogenous jasmonic acid content in tomato flower buds; Figure 1 A: High temperature stress mainly affects the jasmonic acid signaling pathway; Figure 1 B shows that high temperature stress inhibits the endogenous jasmonic acid synthesis pathway in tomato flower buds.

[0041] Figure 2 This is a diagram showing the effect of high temperature on the jasmonic acid biosynthesis pathway.

[0042] Figure 3 This is a diagram of the RNA endogenous competitive network.

[0043] Figure 4 This is a diagram showing the regulation of SlREF expression by SlMYC2.

[0044] Figure 5 To verify the interaction between the promoter sequences of SlLNC402 and SlJAZ7; Figure 5A is a Gus reporter gene experiment demonstrating that SlLNC402 directly binds to the promoter sequence of SlJAZ7; Figure 5 B is a dual luciferase experiment demonstrating that SlLNC402 directly binds to the promoter sequence of SlJAZ7.

[0045] Figure 6 To verify the interaction between SlMYC2 and SlREF promoter sequences; Figure 6 A is a yeast one-hybrid experiment demonstrating that SlMYC2 directly binds to the promoter sequence of SlREF; Figure 6 B is a dual luciferase experiment demonstrating that SlMYC2 directly binds to the promoter sequence of SlREF.

[0046] Figure 7 This is the relative expression level diagram of SlLNC402, SlJAZ7, SlMYC2 and SlREF in tomato flower buds under long-term high temperature treatment. DETAILED DESCRIPTION

[0047] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the following embodiments are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0048] The present invention aims to provide an application of the SlLNC402-SlJAZ7 / SlMYC2-SlREF model in regulating the response of tomato pollen to high temperature stress, including the mining and application of the SlLNC402-SlJAZ7 / SlMYC2-SlREF model, predicting and identifying the SlLNC402-SlJAZ7 / SlMYC2-SlREF thermal response model by using the whole transcriptome data of tomato flower buds under high temperature stress, cloning the full-length sequence of the coding region of SlLNC402 and the coding sequence of the SlMYC2 gene, and verifying the interactive regulatory relationship between SlLNC402-SlJAZ7 and SlMYC2-SlREF by using yeast one-hybrid and dual luciferase experiments. Specifically, it includes the cloning of SlLNC402 coding region, the cloning of SlMYC2 coding region, the verification of the interaction between SlLNC402 sequence and SlJAZ7 promoter sequence, the verification of the interaction between SlMYC2 sequence and SlREF promoter sequence, and the change in the expression level of SlREF in tomato pollen in response to high temperature stress.

[0049] lncRNA long non-coding RNA; circRNA circular non-coding RNA; miRNA micro RNA; JA jasmonic acid; ChIP-Seq chromatin immunoprecipitation sequencing; RNAi RNA interference test.

[0050] 1. Prediction of tomato pollen thermal response model

[0051] like Figure 1 The figure shows the effect of high temperature on the content of endogenous jasmonic acid in tomato flower buds. Based on the expression data of lncRNA, circRNA, miRNA and mRNA of 4mm, 5mm and 6mm tomato flower buds under long-term high temperature treatment, the results show that the differentially expressed genes in flower buds at different developmental stages under long-term high temperature treatment are mainly enriched in secondary metabolic pathways, and are mainly expressed in hormone metabolic pathways with jasmonic acid as the core, such as Figure 1 As shown in A; In addition, it was found that the endogenous jasmonic acid content in tomato flower buds showed a very significant downward trend after long-term high temperature treatment, such as Figure 1 As shown in B.

[0052] In order to further analyze the reasons for the decrease in endogenous jasmonic acid content in tomato buds, it was found that the gene expression levels in the endogenous jasmonic acid synthesis pathway showed a continuous downward trend, indicating that under long-term high temperature treatment, the endogenous jasmonic acid content in the buds decreased, resulting in the inhibition of bud growth. Existing research results show that under heat stress treatment (Heat shock), the endogenous jasmonic acid (JA) content in plants is upregulated, resulting in JAZ protein degradation, releasing MYC2 transcription factors, and activating downstream gene sets that respond to JA and heat stress. However, long-term high temperature treatment inhibits JA synthesis, resulting in insufficient endogenous JA supply, and unable to activate the downstream high temperature response gene set to function. Therefore, it is speculated that there is a new regulatory factor for plant long-term heat response here, which compensates for the function of endogenous JA and promotes the rapid stress response of downstream JA response genes. In addition, SlJAZs all showed a significant decrease under long-term high temperature treatment, such as SlJAZ1, SlJAZ3, SlJAZ4, SlJAZ7, SlJAZ9, SlJAZ10, and SlJAZ11. However, the expression level of SlMYC2 did not change significantly under long-term high temperature treatment. Therefore, it is believed that the endogenous JA content in tomato flower buds decreases under long-term high temperature treatment. There is a regulatory factor that directly regulates the decrease in the expression level of SlJAZs, releases free SlMYC2, and then activates the downstream JA response gene set to achieve tomato heat tolerance, such as Figure 2 Shown is the effect of long-term high temperature treatment on the endogenous jasmonic acid synthesis metabolic pathway in tomato flower buds.

[0053] By integrating the differential expression results of lncRNA, miRNA and circRNA, an endogenous competitive regulatory network of non-coding RNA involved in the high temperature stress response pathway was constructed. In order to explore the regulatory factors that compensate for the function of JA under long-term high temperature treatment, LncTar software was used to predict that SlLNC402 can directly target and bind to the promoter sequences of SlJAZ2 and SlJAZ7, and the expression change trends between SlLNC402 and SlJAZ7 are significantly correlated. Therefore, it is speculated that SlLNC402 can compensate for the lack of endogenous JA under long-term high temperature treatment, release the free SlMYC2 transcription factor by directly regulating the expression of SlJAZ7, and activate the rapid stress response of downstream JA response genes, such as Figure 3 Shown is the endogenous competitive network of lncRNAs upstream of SlJAZs.

[0054] In order to explore the key genes downstream of SlMYC2 that respond to high temperature treatment, referring to the existing SlMYC2-ChIPseq data, it was found that SlMYC2 can directly bind to the promoter sequences of 3389 genes; and referring to the existing SlMYC2-RNAi data, it was found that SlMYC2 can directly affect the normal expression of 2567 genes. In addition, according to the mRNA expression profiles of 4mm, 5mm and 6mm tomato buds under long-term high temperature treatment, a total of 22 differential genes involved in the pollen development process were found, but only SlREF was directly regulated by SlMYC2 under long-term high temperature treatment and was directly involved in the tomato pollen development process. In addition, SlREF is not only significantly upregulated under high temperature treatment, so it is speculated that SlMYC2 positively regulates the expression of SlREF to improve the high temperature tolerance of tomato pollen. Figure 4 Shown is the expression diagram of SlREF regulated by SlMYC2.

[0055] Based on this, a regulatory model for tomato pollen responding to high temperature through SlLNC402-SlJAZ7 / SlMYC2-SlREF was proposed.

[0056] 2. Cloning of the coding sequence of S1LNC402

[0057] Based on the full transcriptome data of tomato flower buds under high temperature stress, the physical location of SlLNC402 on the tomato genome was predicted using stringtie and cufflinks software, and its full length was estimated to be 3817 bp, and the nucleotide sequence is shown in SEQ ID NO. 1. Sequence-specific primers were designed using SnapGene software:

[0058] F: 5'-TGTAGATCTATTTCCCCCCA-3' (SEQ ID NO.5)

[0059] R: 5'-CCTATAACAACACTTCTGAAGTGTG-3' (SEQ ID NO.6)

[0060] The complete DNA sequence of tomato leaves was extracted using Trizol reagent, and the full-length sequence of SlLNC402 was cloned using enzymes using DNA as a template.

[0061] SlLNC402 full length sequence:

[0062] TGTAGATCTATTTCCCCCAATTTCATAAAAATCAAGAGTATTGCCATAAATATTTTAATT

[0063] GTTTTAAAGGGTTCTCATTGTACTGCCAACTCTTCAGCAAATTTCATCAGGCATTTGA

[0064] AGGGTACCTTAAATGGAATTAATGATTACGATTTGTCTATTTATTCATGATAAGCCACAG

[0065] ATTTTAATTTATCCATTTCGCCATGAAAAAGTAGTGTTTTTGGTTTGTCAATTTCGGCATA

[0066] TATGTTTGAGTACAATATGAGTTTTCTTGTTGATAACAACAACAAAGAATATTAATATATA

[0067] GGCTGATGAGGTCGTAGTATAATATGAATGTTTCTCAAAATAGTAGTTGGTGCTCTCTTT

[0068] AAATGCATAGTGCTTATCACTCTGGTTTTGCAGGAACGACCATGATGTTTATATGCTTAT

[0069] GTCGATCTCTTCTTATGCGGATGTTGCTTAACCATTGTTGATGGAGATATGGCTGATCCA

[0070] TTTTTGATTGATGTGGACCAAATGTACTCGCTTTGCCTGCTTTCCCATTTTTTCACTTTA

[0071] GTATTGTGAAGATTAAGTAATTGCAAAAATGTCCTTCTACTCATTTTTATTGTTCTCTGA

[0072] AGTTAAATTCTTAGAAAGCACATTTATTAATTTTGTTACTGCATTTTTCAACAATTAAGA

[0073] CAAATGTATTTTGAATGCTCAATGTTTTTGGACTTTCCAAGGGAATCGGAGAAAGCTTG

[0074] GAATAGTCTTCTCTCTGTTGTTCGTTTTTGGTAATGTAGATTCTCACCTCACCATGTATG

[0075] CATTTTTGAGAAAACGGACATTAAACACATCAACTTTAAGGTTATAGTCCATAGGATTTT

[0076] AGATATAAGTAGTGACAAATGGAGAGGAAAAATAGTGATGTAGTGCTCTTGTTACACTG

[0077] CAGTTCATGTTCATTCCATCATCAAATTCGATGTAAGTTGTTGGAAAGTTTGAAGACCT

[0078] TTCTATAGAGAGCATAAACCTTTGCAATAATGCATTATACTAGCATATATATTCAGTTCAT

[0079] CTGATTGGAAGTATGCTTAAGAACTGTCATTTACTAGTGAGCATATTACTTTGTGGATCA

[0080] GTTATCATCTAGTAAATATAGTTCTTGATCAATGAGGGATAATGTGTAGGGTTTTACTTAT

[0081] CTTATTCGGTTTATGTAGGAGCTTTGATAATAAATAAATTGAAATGTTATAATAACATGTT

[0082] CAGTTTCTTCGTCGTAATTATTTAGTCTATTATGCAAATAACAGTAAATGAAGGTTAGAT

[0083] GTTGATGCATTTTATTTTGTAATTCTTTTGATCCTCTAGGGTTATATTGAGTTTTTAACAC

[0084] ATTTTAGGTGCGTGATAAAGATTTTCGATTAAAATATTTGAGAGTGAAAGAAATGTTTA

[0085] CAGATGATAGAAATTGGGAGAGATTGCTCGTACAGAAATTGCAGAAGTATTGATAACTC

[0086] AATCTGGAAACAGAAAGATAGATGACATCGAGTGTGGAGCTGATTTCTTCAGTTACATT

[0087] CTTCGAAAGGATCTATAATCTTATGCCTGACTAATTTGCGCTCACATCAACATTGCACAT

[0088] TCAAATATATACACTTCTCACAATCTTTGCACCAACCAAGGATTCTAACAAGATTACAAT

[0089] GTTTTAGTCTTGAAATCACAACTACTTATTCATAAGTTCCTGTAGCGCCTGACTAGAAG

[0090] ACTGTAAAAGCCTTTTTGTTGCAATTTCCTATCCATCAGATCTTCTCTGCTGATAATCAA

[0091] TCTAATTTATTTCATATTTTTGCACTTTCAGGAACACTGTAGAGATGTGTAAAATTACATT

[0092] ACTTTGTAGACTGGACCAAAGCCTCCTTGTCGAGCTTGCTAACTTAGTGGAATCTGTCA

[0093] GTTGCATTGGCTAACATGCCAAAGTTATTCAAGGTGATATCTTCAATTTTAACTTGATTG

[0094] ATGTCATCTTTACTCACGCCACTTTGGTAAAACTTTGGAGACGTCTCTCTTAAGAATGA

[0095] TGCACTTTTCCTCTTTAATTCTGGTTTAGAATGGATATTCAGAAGTCAAACAATTTGCTG

[0096] AAATTGAAGTTTTGGTTTGTGTTCTCAAATATCTCATTTGATTATTTACCCTTTTCCTAG

[0097] CCAACAATTTTCAGGAAAAATATCAACAGAAGATAACTATTATTGAGCCTATCGAATCAT

[0098] TTATTGCAATAGCTGCTACATGGAAGTCTTTCTTGTTATTTGCAGCAGCAGCAAAACATGTAC

[0099] AAGCAATAGACAGTTCTCGAGCATATATTCTTTTCCATGAAAGCGAAAGCACGACATGA

[0100] AATTTGAGACTACTTTAATGGTATTATAGATTTTATTAAGAGTTCTTGGTTATATTCTTC

[0101] AAAGCCACGAAGCCTTCAATCTGAGGTCAAGCCTTTTTTTAATTGACGAAGACTCCCTG

[0102] GATTAATGGTCTCTCAAGAGGTCACACAAGGTAAACTGATTCAGACATCCTTCTACAT

[0103] ACTATGCTAGTACCATTTAATTGAAGTTCCTCACTGGCCAGTTCAATTGTTTTATGTCAA

[0104] TGTGACTATAGAAGGTGAAAATCGAGCTGTTATTTCTAACCACAAGTTAGAAACTAAAT

[0105] TCGGACAATCTCATGTTTGTTTTGTATCAACACTGCTAGAAAAATGGTAGGACTCTTAAA

[0106] ATTGCTTGTACAACTTCTCTACTAAAAGTAGTTCATTTATATAATAAGTTGTTGCTATGAA

[0107] GATGAAACAGAATGGGGAAAAGAGATACTAATTCTGATTCATCTCTTAAAATTCCTCGC

[0108] TTTCAAATCGTGTTCAAAATGTAGTGATTGTTCAATCTAACTACTTTGAAAATGCAGATT

[0109] GGCTAGATATATGGTTAGGTCATTGAGGACATATTAACAGGCTTCAAAATGCAATGTCAT

[0110] GGATGGAGATCCATATATTGTATGCTTGTGTGATGAGTGCTGCTGCTGCAGCTGCAATGT

[0111] TTCCTTAGGAGTTTTGGTATGTCCATGATATGTTGAACTGGTCTGTAAGCTTTCCTCTTC

[0112] TGAGTCTGCCATTCTTCATCAGCTGGCTTGGTGCTTCCTTGTTTTTTTGGGTCTGTCCATGC

[0113] TATGTTATTTCTATGTTTTGATGTTGCTGGTATTGTGAACTGGTGTTTCCAACCTCTCTAT

[0114] CTGTGTATTGCATTTGCAAGGTTTCTGGAATCTTGTTTGACTTGTTCCCTTTTGTTTTTG

[0115] TTCTTCCTATTAGCCTCAAACTCTTTTCTTTTTTCAAGTTCCGCATCTCTTTGCTTAATAT

[0116] TACATAATGTAACAAGTGTCCTTCTTGTTTACAATACATGTGGAAATCAGAATTCATGTG

[0117] CCGATAAATGTTATATATATATATATATATGTATGTATGTATATATATATATATATATATGTATAT

[0118] TGGTTGTCTTACAATGAATACTCTCCAATTATGTACTTTTACATTTATTTCACTAACTTGT

[0119] AATTTCATTTTGAATGTGCAGAAAAATGCCAGCAATTGCATTTGAATTGTAGCCAGAAT

[0120] TAAACTCGGTCTATTGTAGTGAACGATGAAGAGTTTTTGTACTATTGTCATATATGTTCG

[0121] GATGCATTTGGTTTTGTCGAATTTATTGATAGTAGAGATCTTTAAGCAATCACTTTCTTA

[0122] AGTATTGGTTGTACACGACATATTTCTCAAAGTTTATTTAAAATACATAACTTTGATATAA

[0123] TTATTAGTTAATTTTGTGTTTTAGGTTCCTTTGTATATTTAATTATGTGACTATTGAATATAT

[0124] GCAAATAAAAATTATTAAGCTTAAAAATAAAAAATTGTAGCTACAAAATTCTACACAAC

[0125] ATTGCCACACTTCAGAAGTGTTGTTATAGG

[0126] 3. Cloning of the coding sequence of SlMYC2

[0127] The nucleotide sequence of the SlMYC2 coding region is shown in SEQ ID NO.2, and sequence-specific primers were designed using SnapGene software:

[0128] F: 5'-ATGACTGAATACAGCTTGC-3' (SEQ ID NO.7)

[0129] R: 5'-TTAGTGTGTTTCAGCAATTTTC-3' (SEQ ID NO.8)

[0130] The extraction of tomato total RNA was carried out according to the instructions of Trizol extraction reagent (Takara), and the first-strand cDNA synthesis was carried out according to the reverse transcription kit PrimerScript TMII The 1st Strand cDNA Synthesis Kit (Takara) was used as the instruction. Tomato cDNA was used as the template and Primer STAR Max high-fidelity enzyme was used for amplification (Takara). The annealing temperature was 52°C.

[0131] SlMYC2 coding region full length sequence:

[0132] ATGACTGAATACAGCTTGCCCACCATGAATTTGTGGAACAATAGTACTAGCGATGATAA

[0133] CGTTTCTATGATGGAAGCTTTTATGTCTTCTGATCTTTCTTTTTGGGCTACTAATAATTCT

[0134] ACTTCTGCTGCTGTGGTTGGTGTCAATTCAAATCTTCCTCATGCTAGTAGTAATACTCCC

[0135] TCGTTTTTGCACCATCTTCTTCTACATCTGCATCTACTTTATCCGCAGCTGCGACTGTG

[0136] GATGCTTCCAAATCTATGCCGTTTTTCAACCAAGAAACCCTTCAGCAGCGTCTTCAAGC

[0137] TCTTATTGATGGTGCTAGAGAGACGTGGACTTATGCTATCTTTTGGCAATCGTCGGTTGT

[0138] TGATTTCTCAAGTCCGTCTGTGTTGGGTTGGGGAGATGGTTATTACAAAGGGGAAGAA

[0139] GATAAAGCAAAAAGGAAATTATCGGTGTCATCACCTGCTTATATTGCTGAGCAGGAGCA

[0140] TCGGAAGAAGGTTCTACGGGAGCTGAATTCGTTGATTTCCGGGGCACCACCCGGAACG

[0141] GATGATGCGGTTGATGAAGAAGTTACCGACCCGAATGGTTCTTTCTTATCTCCATGAC

[0142] CCAATCGTTTGTTAATGGAAGTGGGCTTCCTGGTCAGGCGTTGTATAGTTCCAGCCCGA

[0143] TTTGGGTCGCCGGAACTGAGAAATTGGCAGCTTCACACTGTGAACGTGTGAGGCAAG

[0144] CACAAGGGTTCGGGCTTCAGACGATTGTCTGTATTCCTTCAGCTAACGGCGTGGTTGAA

[0145] TTGGGCTCGACGGAGTTGATTGTTCAAAGTTCTGATCTTATGAACAAGGTTAGAGTATT

[0146] GTTTAACTTCAGTAATGATTTGGGTTCTGGTTCATGGGCTGTGCAGCCGGAGAGCGACC

[0147] CATCGGCGCTCTGGCTCACTGATCCATCGTCCTCAGGTATGGAAGTTAGAGAGTCTTTA

[0148] AATACAGTTCAAACAAATTCAGTTCCATCTAGTAATAGTAATAAGCAAATTGCTTATGGA

[0149] AATGAGAATAATCATCCATCTGGAAATGGTCAGAGTTGTTACAATCAGCAACAACAGA

[0150] AGAATCCTCCTCAGCAACAAACACAAGGATTCTTCACGAGGGAGTTGAATTTTTCGGA

[0151] ATTCGGTTTCGATGGAAGTAGTAATAGGAATGGAAATTCATCGGTTTCTTGCAAGCCTG

[0152] AATCAGGAGAAATCTTGAATTTTGGTGATAGTACTAAAAAAAGTGCTTCCAGTGCCAAT

[0153] GTGAACTTGTTTACAGGTCAGTCCCAATTTGGGGCTGGGGAGGAGAATAATAACAAGA

[0154] ACAAGAAAAGATCAGCTATCCAGGGGAAGCAATGAAGAAGGAATGCTTTCATTTGT

[0155] TTCAGGTACAGTTTTGCCTTCTTCGGGCATGAAGTCAGGTGGAGGGCGGAGGCGAAGAC

[0156] TCTGAACATTCAGATCTCGAGGCTTCAGTGGTGAAAGAAGCTGATAGTAGTAGAGTGG

[0157] TAGAGCCTGAAGGAGGCCAAGGAAGCGAGGTAGAAAGCCAGCGAATGGACGGGAG

[0158] GAGCCATTGAATCACGTCGAGGCAGAGAGGCAAAGGAGGGAGAAATTGAACCAAAG

[0159] ATTTCTACGCGCTTAGAGCTGTTGTACCAAATGTGTCTAAGATGGACAAGGCATCACTCC

[0160] TTGGGATGCTATTTCCTATATAAACGAGTTGAAATCGAAGCTTCAAAATACAGAGTCA

[0161] GATAAAGAAGACTTGAAGAGCCAAATAGAAGATTTAAAGAAAGAATCAAGGCGCCCC

[0162] GGTCCTCCTCCACCACCAAATCAAGATCTCAAGATGTCTAGCCACACTGGAGGCAAGA

[0163] TTGTAGACGTGGATATAGACGTTAAGATCATCGGATGGGATGCAATGATTCGTATACAAT

[0164] GTAATAAAAAGAATCATCCAGCCGCAAGGCTAATGGCAGCGCTCATGGAATTAGACCT

[0165] AGACGTGCATCATGCCAGTGTTTCAGTTGTCAACGATTTGATGATCCAACAAGCCACA

[0166] GTGAAAATGGGTAGCAGACATTACACTGAAGAGCAGCTTAGGGTAGCGTTGACATCGA

[0167] AAATTGCTGAAACACACTAA

[0168] Primer STAR Max high-fidelity enzyme amplification system:

[0169]

[0170] Primer STAR Max high-fidelity enzyme amplification conditions (three-step amplification):

[0171] Step 1: 98℃ 10sec

[0172] Step 2: 55℃ 5sec or 15sec

[0173] Step 3: 72℃ 30~60sec

[0174] Repeat 30 to 35 times.

[0175] 4. Cloning of promoter sequences of SlJAZ7 and SlREF

[0176] According to the physical position of SlJAZ7 on the genome, the sequence 3000 bp upstream of its transcription start site was extracted as the promoter sequence, and the nucleotide sequence is shown in SEQ ID NO. 3. Sequence-specific primers were designed using SnapGene software:

[0177] F: 5'-ATGTATAACAAATTATACAGTTTTATGT-3' (SEQ ID NO.9)

[0178] R: 5'-ACAAAATTTATGGAAGATAAAAATGA-3' (SEQ ID NO.10)

[0179] The promoter sequence of SlJAZ7 was cloned using DNA polymerase using DNA from tomato leaves as a template.

[0180] Promoter sequence of SlJAZ7:

[0181] ATGTATAACAAATTATACAGTTTTATGTTTGCTATGGAGCGCAATTATGCAAAGTTTGCTA

[0182] TAGCATACAAATATGAATTTTTTATTTGCTATATGTAAAAGTTGTCCAATATTTAAACTATT

[0183] TTAAAAATTCATTACTGTCAAATGTGAAATCAAAATACATATTTAACATAACAAATAAAT

[0184] AAGTATTGGAACAATTGTATTATTAGAAAAATAAGAAATGAGCCATGTACCAAATTTCCT

[0185] AGGTAATTAACAAATCTAGTTTAACAAAATTCATCAACAAATTCCATAGCAAATTTTATG

[0186] TTTTCTAATTATGATATATTATAAATTTGTAAATATATATATATATATATATATATTCACATAAA

[0187] TGATCAAAGAACGTTTCAGTTCATTAATTCAAATTTGTAAATATATACTTGTAAAAAATA

[0188] CTCAAAGAACATTTCAATTAATTAATTTAATTTCATATCATACTTATATTATTTTGTAAAAAA

[0189] ATTTAGCTGTCATTCTATACGTTCATTGGTGGTGCACAAGTCATAAAAAGAAAAAAAAT

[0190] GTGGAAAATCTTAGAAAAATTAAAAACTTAAATTATCTAATATATATATTATAGACTATAT

[0191] ATAGTACCTAGTGGCATATAATGTTAATTTTTTTTTTGCTAAATATGATAAACATGGATAA

[0192] AAGACTAAGTCATTTCTCACTATTGAATTAATGAAATGAATAATTACAAAAGGACCATG

[0193] TCAATTTAGTTTACATTGGAAAAAAGTATCTTTGGTAGACTTATGAAATATTAAAAACTA

[0194] ATTAAAAATAATTTAAACATGCTTAGGATAGAAACTACCCCTACTTCTCCTTAATACTAA

[0195] AAATTAATCCACTTTTTGTTTTCACTTCAAACTATTTTATATGATTAAATTTGACTGAGGGA

[0196] TATATTTTCTTTACATGACATCAAAGTTAGATTCACATTATAATTATTAATATATCTGACAT

[0197] TAATCGATCTTCATTATGTTTCTCATGTTAGATCATATTTTGACCACATCAATTGAAA

[0198] CGTGAATATGTGACTCATTTTATTATATGATTCAATCGTTCCTCACATCATGAACAAACTTT

[0199] TGTGTATAGTAAGACTAAACGTCCATTTTCTTAACAATTAGTTCATAAAAAAATTATTTT

[0200] TTCTTTTTAACATTTCGTATGCTGGTTATATTAGTATAACATGGTTAAAATAGTGACTTTC

[0201] ACCTCAAATGACTCTTTTTTTTTTAAAAAAAAAAACACTATGAAAGTATGTACATGCTAT

[0202] GTCAAGTACGAAGTATAGAACATTTAATTCAGGACCACCGTTATATAATAAAATATATAA

[0203] CAATGTAAATTACTATATCAAATAAGAAGTGTAGAACATTTAATTCAGGACCACCGTAAT

[0204] ATAATAAAAATGTAAACCTATAGCTATAGAAGGTTAAATAATGACATTAATAGTATATGTG

[0205] ACCTTTGGGATGCCAATAGGACATTGAATAAGATGTAAGTACCTTCCTCGTTTTAAACAT

[0206] TTTTATGAGACGTAAAAATTATAATATCAATAATATTGTGTCTCAATTTAAAATATATTGAT

[0207] ATTAATATACAAATTAATATGTATCTAATAAAGATATTTATGTAATTTTTTTACATAAGACC

[0208] TAAATGTTAATTTATTATATTAATTTGAGCTTATTGTAAAATTATAAATTTCACAATTTGAA

[0209] AAAAAAAAAAACTTAGTAGTAGTAGGTAATAGACAACTATAATGGATAAATAACTCAAC

[0210] TATTCTCACCAATAATAGATGAGGCCACATAAGGAACACGTCGTCGTTTTAGTTCACATT

[0211] GGATATTTGGAGCAAGTAATATCTTTATAGACAATTGTGAGTTTGATTTTTGATCTTTCAA

[0212] TTTGATATCATTATTAGAATTTGATTTATTTTAAGTTACAGTGTATAAAAAAATTTATGCT

[0213] AAAATGTTTCTAAATCTCTGATTAAGAAATGAACAATTCCATATAAATAATTCGATTTTAA

[0214] AGGAAAATACATCAAATTATTTTGTATATATCTTTTGTTTTAAACTCAAACTCGATTAATT

[0215] TAAACACATGTTACAAGTTCATGCGAATATTTTTCAGCCCCCCATAAAGGTATTTCCT

[0216] TTTTGTGGTCAAACTCGAAAGTTCTGAGAAATAACAAAGAAACTTTTCACAAAGATAA

[0217] TCGCAGAACGTATCATCAATCACCACCATAATATTTAATACATCATACCAATATAAGTTGT

[0218] GGTGCAGGTGAGGCTAGTTCGCTCTAAATTAGAAGTTTTGGCCTTTGCGTATGAAGAAA

[0219] TTTCTATTGGGAGAGTGCTATCTCTGAATAGATCCTGTAATGTGCGATCTAAATTTAATCT

[0220] AAACTTTAATATGGACTTGTAACACTGAATAAGAAATAAATAAAAACCTAAAATATAGTT

[0221] ATTAGTACTAGAAATTTTCATTTTATAAAACAAACAATTATAATTACGTTACGTAGT

[0222] ACATGTGGCCTTTGAGATAAAAATAGGACAATTTGATTAGACATAAATACATTCCCTCGT

[0223] TTTAAACATTTCTCTAAAATATTATTATATTATTTTCTTTATTTTAATTTATTTATTCTAATTT

[0224] AATTTAAAAATGAAATTTAGAAAATAAGAAAGTCTTTTAAATTTTATGGTATTAAAACAT

[0225] GTTAGGTAGAGTTTGAAATCAAAAAGTCATAAAAAAAAATCAACCCTTTTTTTAAATAA

[0226] ATTAAAAAAGAAAAAAGAAAAACAGATAAATTAAAACGAAAAAGAACTATTTAATTAA

[0227] ATATCAACCAATCATTTTATTTTTGTATTTCAAATCAATTAAAACCTGCACTTAACCCAA

[0228] AAATAAAAAAACAAGTTGTGGCTATCAATTTAGAGGGACCAATTCAATAAACACGTTTT

[0229] TTTTCTGTCTAATTTATTTTATTTTATTTTTTTAGAGAATTTTAATAGTTGTTTCTTCTTCTT

[0230] TTTCTTTCATTTTTATCTTCCATAAATTTTGT

[0231] Full-length sequence of SlJAZ7 coding region (SEQ ID NO.21):

[0232] ATGGATTCAAGAATGGAGATAGATTTTATGGACCTCAACAGCAAACCAAAATTATCAGA

[0233] AATGGAGAAGCAACACAAAAAAGTATCTGGAATGAAGTGGCCATTTTCATTGGCTGAT

[0234] TTAGCTACTCACCATGAACACACATTTTTTCAGAATTACAAATCCACCCCAATAGTTTCC

[0235] ATTAATTCAAAAAATTCATCCCTAAACAATTACAAATCCACCATTGACCCCCAATACTTT

[0236] AGAGGGACTTTTCCTCTATTAGCAAAAACAAGCACTTATGACTCAAGGAAAAATTATG

[0237] ACAATTTGAGTCCAAATGAGTCAACATTGACCATATTCTACATGGGTGAGGTCCATATTT

[0238] TTCCGGGTATATCACCAGAAAAGGCTGAGCTTATAATTGACCTGGTTTCTAAATCAACA

[0239] ACTCTCCACATGGATGAGATTTTAGAAAAAGTGATGAATAAAGAAAAATATGAAGAAA

[0240] ATAAATCAGACCCTTCAAATGCATCCACAAATTATGCTAAAGGAGCACTTGCTATGGCT

[0241] CGTAGAGCAACTCTTGCACGATTTTTGGAGAAGAGAAAACATAGATTGATCAAAGCTA

[0242] GGCCGTATCTATATGGGGAAAATTTATCAAAGTTTCCCTTTGATATTCAACAACAAGAA

[0243] GAAGAAACGGCGTCGTCAAGCGTTCATTGGGAAAACTAA

[0244] According to the physical position of SlREF on the genome, the sequence 3000 bp upstream of its transcription start site was extracted as the promoter sequence, and the nucleotide sequence is shown in SEQ ID NO. 4. Sequence-specific primers were designed using SnapGene software:

[0245] F: 5'-ACTTACATCACTAAGATGCAAAAG-3' (SEQ ID NO.11)

[0246] R: 5'-ATTCTGCTGGTTGTATGGAAG-3' (SEQ ID NO.12)

[0247] The promoter sequence of SlREF was cloned using DNA from tomato leaves as a template and DNA polymerase.

[0248] Promoter sequence of SlREF:

[0249] CTTTGTTTCATGCTCTCCGATGAATTATGTAAGTGTTGCAAAACTCCAAAGTAAACTG

[0250] GAAGGAGAAGCCAACGAAATTGCGGTGAAGAAGCTAATTGATAAAATGACAGAAGAT

[0251] GGATAATTTGAGTCTAAGGGCTACCGCAGACTAGGTACCCTCAGGCCTCTGGCAAACA

[0252] GTATTTCCTGCTTTGTATAGAGCAAAACGAAGTACAACTTTCCCAATGATGGACTGATT

[0253] TGGTCATTGCAGGGAAGCGTGTGATCCACTCTGATCTGACTGAAGAAAAGCTGGCTGA

[0254] AGTTACGAAGGTCTTAGCAAAAGACAGTATGGTAATAGTCTTGAATTAATATAACTTCG

[0255] ACTGTTTCTTGCACTTCTATTTTAATTCTCATCTTAAATTATTATAAATGGTCAGGATATTG

[0256] ACATGCATGAATCAGGTAATGATTCCACCTATCTGAATTTTGATATCGTTTAGTTAAGTTA

[0257] CTTCTACACTAATCATCAAAGATCAGACCACAAAATCTAATATTACTGTATGGCTACTAG

[0258] ACAACAACTGTAGATATCTATCCACTTGTGGTGCTCTCCAGTCTATCGGATCGGATCTCA

[0259] CACGTACCAGAGGCAAATGTGATGCATATTGGAACGGTTCTGTCATGAGCGATCAGAC

[0260] TGTCTCCAAGAGAAAGGAGCATGAAAAAACACCATCAAGCAACGCTGAGGTTGGTTC

[0261] AGTTGTGGATTATCTCAATAATCTTCATTAACAATGTAACTCAGTAATTAATTAGTTGATT

[0262] ATCGCCTAACAAAAGTGCAGTAACAAATAGTTAGATATATATGATGTTTGTGCAGCCTGT

[0263] AGCTTCTCGGGAGAGCTTTGTTCCTGGTAAGGAGAATGGAAGGAAAACCAAACTAAC

[0264] CTGATGAGTATGACATTGTCTGTAGTAGGTCCAGTCAGGAGAAACGTCAGAGGAAAGC

[0265] AAGCATGGTACGATATCAGTCTTTTAGAAAATCTTAGTAAAGAATCCTGGCTGAAGAAG

[0266] TATTTGGGAAATATTTCTAATTGTTCTTCTCTTCTCTTTTCTCTATATACAGGTAAAAGAA

[0267] CCTATTCCAGTATGTTAGACGCCAAACCTCAAGTTGCTTGATGGTTCCTTAACTTGAG

[0268] TGGTGAAAAATCTAGCAAGTTTCCTTACTCTAATAGTTGTACTCAAGTAACAACATAGA

[0269] TCGATGCTATAAATAGGTGATATTTTACCTTTTCTTGATGGTGCCACACTTGCTTCTGGCTT

[0270] CTTCATTACTTTTATTTTATGATGTACCGACTGATTTGTAATAGATGTGACGTGACCACGA

[0271] TTCAAATGCTCCCTGTTGATATAAATATATACTAGCTTCTTGGTACATTGATTTTGCCTGT

[0272] TCTAAGAATAAACGAAAATGTTCCCTACGATACATTTATTTTTCACATTAATATAACAGT

[0273] GGCTTAAAATAAAACGTGACCAATTTTTCGGAGTATACTTTTTGTCTAATTGGTATGAAC

[0274] TTCTAAAAGAAATACTAATTGAAAAAGGAAGCAGGTATAGATAGAGAATAATTAATTAT

[0275] TAGTATGACGCACCTGTAAGATGTCTTAATTGTATAGTGTAACAGCAAGGTGGAACGGT

[0276] AATTTTACATTTCTCTGCCTTATCTTAGATTTCAAGATTTAGTAATTAATAATTTCTCTCTT

[0277] TTTGAGTACTACACTACGCCCACAGTTCACAAACAAAAAGGACAAAGTTGGAAAA

[0278] TAAGCGTAAAAGGTCGAGATATTTCGCACTGCCTTCAAACAGAAATGGCACGTGTCCT

[0279] TCTCGAAGCCTCCACACACCCCTAGAAATGGTCATTTCCTCAAACGCCTCAATGATTCT

[0280] GGACTAAGGAATCCCTCCTGACCCCATAGTAACGACACGTGTCATCTTCACAGCCCTTT

[0281] GATTTTAATTAAATATTCCCCCACTTCCTTTATCACCCAACAAATTTTCTCATTTGCATCA

[0282] CCAACAGCTGAGCAAATCAAACTTCCATACAACCAGCAGAAT

[0283] Full-length sequence of the SlREF coding region (SEQ ID NO.22):

[0284] ATGTCTACCAACAAGGTTGAGATGGAGAAATGCGAGGTTAATTTGAAACATCTAGGGT

[0285] TTGTAAGGGTGTTGGCTCTAAACACTGCAGTTTTGTTGTCAAATTTGTATGATTATGCGA

[0286] AGCAGAACTCGGGGCCTCTGAGATCAACTGTTGGTACAGTAGAAAACGCTGTAACCAC

[0287] CGTCGTAAGGCCTGTTTACGATAGATTTAAAGGTGTTCCTGATGAAGTCCTTGTTTTCCT

[0288] AGACAAAAAGTTGGATGATGGAACAGCAAAATTTGATGAACATGCTCCTCCATTGGCC

[0289] AAGAAGGTTGTCAGCAGTGTCCAGTCTTTGTTTCAGAAGGCATCAGAGGTAGCACAA

[0290] GGCTTGTCCAAAGACATACAGGAGGGAGGTCCTCGTGCAGCTATCTATCATGCTGGTG

[0291] AATTGTCTAAGCAATTTGGTACTACGCAAGTGGCAGTACTCTGGTATCATGTCAATCATT

[0292] GTCCACCATTGCACGGAATCGCACAGATGGCTGCTCCTACAGTTGCTCACTGGTCCGA

[0293] AAAGTACAATCATTTGGTAGCTAACTTGCAAGAGAAGGGTTATAGTGTTGTCAGCTATA

[0294] TCCCTTTGATACCTGTTGAAGAAATCTCAAAGGCATATAAACAGGTTGAATCTGCTGCA

[0295] ACGAAGAAAGAAGGTGCCACTGACTCGACCTCTAGTAAATCTGAATGA

[0296] 5. Verification of upstream and downstream interactions of the regulatory model

[0297] By integrating the differential expression results of lncRNA, miRNA and circRNA and the endogenous competitive RNA network, we predicted that SlLNC402 targeted the interaction region of SlJAZ7 promoter. We used the dual fluorescence molecular complementation system to transiently co-express nYFP and cYFP fusion proteins in tobacco and found that SlLNC402 could target the SlJAZ7 promoter region and regulate its expression. Figure 5 As shown in A. In addition, through the dual luciferase test, the SlLNC402 full-length sequence vector and the SlJAZ7 promoter sequence vector were constructed and co-transfected into tobacco, and it was found that the combination of the two activated the expression of the β-glucuronidase gene, and the leaves appeared blue after being stained, as shown in FIG. Figure 5 As shown in B. The above results indicate that SlLNC402 directly binds to the SlJAZ7 promoter sequence and positively regulates the expression of SlJAZ7.

[0298] The JAZs-MYC2 module is a core conserved step in the JA signal transduction process. Using the previous SlMYC2-RNAi and SlMYC2-ChIPSeq data analysis, it was found that SlMYC2 targets SlREF, and SlREF is significantly upregulated in tomato flower buds under long-term high temperature stress. Using yeast one-hybrid and dual luciferase interaction experiments, it was found that SlMYC2 targets the promoter sequence of SlREF, such as Figure 6 shown. Figure 6 A is a yeast one-hybrid experiment, demonstrating that SlMYC2 directly binds to the promoter sequence of SlREF; Figure 6 B is a dual luciferase experiment, with the ordinate representing fluorescence intensity, and the results show that SlMYC2 directly binds to the promoter sequence of SlREF.

[0299] 6. Verification of the expression trend of the SlLNC402-SlJAZ7 / SlMYC2-SlREF model in thermal adaptation of tomato flower buds

[0300] According to the coding region sequences of S1LNC402, S1JAZ7, S1MYC2 and S1REF, sequence-specific primers were designed using the Primer-BLAST function of the National Center for Biotechnology Information website.

[0301] SlLNC402-F: 5'-TCTTCAAAGCCACGAAGCCT-3' (SEQ ID NO.13)

[0302] SlLNC402-R: 5'-GCAGCACTCATCACACAAGC-3' (SEQ ID NO. 14)

[0303] SlJAZ7-F: 5'-TGGCTCGTAGAGCAACTCTTG-3' (SEQ ID NO.15)

[0304] SlJAZ7-R: 5'-CCCAATGAACGCTTGACGAC-3' (SEQ ID NO.16)

[0305] SlMYC2-F: 5'-CAGTTTTGCCTTCTTCGGGC-3' (SEQ ID NO.17)

[0306] SlMYC2-R: 5'-TTCGCTGGCTTTCTACCTCG-3' (SEQ ID NO.18)

[0307] SlREF-F: 5'-TGTAACCACCGTCGTAAGGC-3' (SEQ ID NO.19)

[0308] SlREF-R: 5'-ACCAGAGTACTGCCACTTGC-3' (SEQ ID NO.20)

[0309] The extraction of tomato total RNA was carried out according to the instructions of Trizol extraction reagent (Takara), and the first-strand cDNA synthesis was carried out according to the reverse transcription kit PrimerScript TMII The cDNA of tomato was used as template and PrimeScript was used to synthesize the cDNA. TM RT-PCR Kits amplification (Takara), annealing temperature was 60 °C.

[0310] The results showed that the expression levels of SlLNC402 and SlJAZ7 decreased significantly in flower buds at different developmental stages under long-term high temperature treatment. Figure 7 A and 7B. The expression level of SlMYC2 showed a trend of gradual increase with the development of flower buds, but the effect of long-term high temperature treatment on its expression level was not significant. Figure 7 C. In addition, the expression level of SlREF was dramatically upregulated after long-term high temperature treatment, as shown in Figure 7 As shown in D.

[0311] In summary, it was shown that SlLNC402 directly regulated the decrease of SlJAZ7 expression, released free SlMYC2 and activated downstream SlREF, which positively regulated the heat stress resistance of tomato pollen or plants.

[0312] The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in this field.

[0313] Finally, it should be noted that the above description is only a preferred example of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of the SlLNC402-SlJAZ7 / SlMYC2-SlREF model in regulating tomato pollen response to high temperature stress, characterized in that: The SlLNC402-SlJAZ7 / SlMYC2-SlREF model includes SlLNC402, SlMYC2 gene, the promoter of SlJAZ7 gene and the promoter of SlREF gene; The SlLNC402-SlJAZ7 / SlMYC2-SlREF model is positively regulated in tomato pollen response to high temperature stress; The nucleotide sequence of S1LNC402 is shown in SEQ ID NO.1; The nucleotide sequence of the S1MYC2 gene is shown in SEQ ID NO.2; The promoter nucleotide sequence of the SlJAZ7 gene is shown in SEQ ID NO.3; The promoter nucleotide sequence of the SlREF gene is shown in SEQ ID NO.

4.

2. The use according to claim 1, characterized in that: The SlLNC402 positively regulates the expression of the SlJAZ7 gene and negatively regulates the response of tomato pollen to high temperature stress; The SlMYC2 positively regulates the expression of the SlREF gene and positively regulates the response of tomato pollen to high temperature stress.

3. The use according to claim 1, characterized in that: The promoters of the SlLNC402 and SlJAZ7 genes positively interact; The promoters of the SlMYC2 and SlREF genes positively interact.

4. The use according to claim 1, characterized in that: The primers for amplifying S1LNC402 are shown in SEQ ID NO.5 and SEQ ID NO.

6.

5. The use according to claim 1, characterized in that: The primers for amplifying the SlMYC2 gene are shown in SEQ ID NO.7 and SEQ ID NO.

8.

6. The use according to claim 1, characterized in that: The primers for amplifying the promoter of the SlJAZ7 gene are shown in SEQ ID NO.9 and SEQ ID NO.

10.

7. The use according to claim 1, characterized in that: The primers for amplifying the promoter of the SIREF gene are shown in SEQ ID NO.11 and SEQ ID NO.

12.

8. A method for improving the response of tomato pollen to high temperature stress, characterized in that: Under high temperature stress, the expression of SlLNC402 was reduced, thereby reducing the expression of the SlJAZ7 promoter, thereby relieving the inhibition of the SlMYC2 gene, increasing the expression of SlREF, and ultimately improving the high temperature resistance of tomatoes; The nucleotide sequence of S1LNC402 is shown in SEQ ID NO.1; The nucleotide sequence of the SlMYC2 gene is shown in SEQ ID NO.

2.

9. The method for improving the response of tomato pollen to high temperature stress according to claim 7, characterized in that: The primers for S1LNC402 fluorescence quantification are shown in SEQ ID NO.13 and SEQ ID NO.14; The primers for fluorescence quantification of the SlJAZ7 gene are shown in SEQ ID NO.15 and SEQ ID NO.16; The primers for fluorescence quantification of the S1MYC2 gene are shown in SEQ ID NO.17 and SEQ ID NO.18; The primers for fluorescence quantification of the SlREF gene are shown in SEQ ID NO.19 and SEQ ID NO.20.

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