Cloning,expression And Functional Analysis Of SlMYB13 Gene In Tomato

Soil salinization is the main reason to inhibit agricultural production.It is of great significance to cultivate saline-alkali tolerant plants for rational development and control of saline-alkali land.MYB transcription factor plays a key role in stress response.Further study on the role of MYB transcription factor in plant stress response is conducive to exploring new members of MYB gene family that may be involved in plant abiotic stress regulation.Tomato is an important vegetable crop in the world and an ideal model plant.It is of great theoretical and practical value to study the mechanism of MYB gene in tomato stress tolerance.In this study,a MYB transcription factor named Sl MYB13 was isolated and cloned from tomato leaves.The tomato was genetically transformed by expressing,positioning and constructing an overexpression and gene editing vector to verify the function of Sl MYB13;And that action mechanism is study through RNA-seq analysis.The research contents are as follows:1.Cloning and bioinformatics analysis of Sl MYB13 gene.Sl MYB13 is an R2R3 type MYB transcription factor.The number of amino acids was 255,among which Serine（Ser）content was the highest,with 29 Serines accounting for 11.4%.Methionine（Met）and phenylalanine（Phe）were the least abundant,with 4 of them accounting for 1.6%.It does not contain pyrrolysine（Pyl）or selenocysteine（Sec）.Molecular formula C₁₂₇₉H₂₀₀₁N₃₆₉O₄₁₆S₉,total atomic number 4074,molecular mass 29491.72 Dka.The theoretical isoelectric point is 5.16,the total number of negatively charged residues is 41,and the total number of positively charged residues is 30.The high mean value of hydrophilic protein-0.870 is hydrophilic protein.The instability index was 57.69,indicating that the protein was unstable,and the aliphatic index was 76.47.The amino acid sequences of Sl MYB13 and homologous genes were analyzed.Among them,tomato MYB13 was closely related to the wild Pannali tomato MYB13,potato MYB13 and wild diploid potato MYB13,which could be classified into the same group.The analysis of Sl MYB13 promoter showed that ABRE plays a role in ABA response,CGTCA-motif and TGACG-moti play a role in MEJA response,and DRE and TC-rich repeats play a role in plant stress response.2.The tissue expression pattern of Sl MYB13 gene was analyzed.The expression of Sl MYB13 was the highest in the root,followed by that in the flower.The expression level in the stem and leaf was similar,the stem was slightly higher than the leaf,and the expression level in the fruit was the lowest.In addition,analysis of salt and drought expression patterns indicated that the Sl MYB13 gene responded to salt and drought stress.Under salt stress,the expression of Sl MYB13 was increased at the first 12 h,and then decreased significantly to 1/6 of that of the control plant 24 h later.Under drought stress,the expression of Sl MYB13 mainly showed a downward trend.After treatment for 1 h,the expression was about half of that of the control;after 24 h,it was significantly lower than that of the control plant and the expression was about 1/7 of that of the control.3.The construction of the BWA（V）HS-Sl MYB13-GLosgfp expression vector and transient subcellular localization through the epidermis of tobacco leaves indicated that Sl MYB13 was localized in the nucleus.4.The OE plants overexpressing Slmyb13 and the MYB13-knockout plants were obtained by transgene and gene editing techniques.The germination experiments of T3generation seeds in Na Cl solution（50 m M and 60 m M）were carried out.The results showed that the germination rates of myb13 plants（80%and 60%）were higher than those of OE lines（30%and 0%）and WT plants（40%and 40%）.The T3 generation seeds were treated with simulated drought stress using PEG6000 solution（5%and 10%）,and the results showed that the germination rates（80%and 60%）of mutant myb13 seeds were higher than WT（70%and 50%）,while the germination rates（50%and 40%）of OE seeds were lower than WT.The appearance of both treatments was myb13>WT>OE.The T3generation plants were subjected to salt stress and drought stress in the five-leaf and one-heart stage.The results showed that compared with WT wild-type plants,OE plants exhibited serious wilting,while myb13 plants grew well.The results of CAT,POD and SOD activities showed that each index of myb13 plant was significantly higher than that of WT control plant,and the overexpressed OE plant was significantly lower than that of WT plant.The proline content of myb13 plants was significantly higher than that of WT control plants,while the overexpressed OE plants were significantly lower than that of the control plants.The MDA content of myb13 plants was significantly lower than that of WT control plants,while the overexpressed OE plants were significantly higher than the control plants,which indicated that Sl MYB13 negatively regulated the salt tolerance and drought tolerance of tomato.5.The transcriptome of tomato leaves after 5 days of salt stress was sequenced.The expression levels of differential genes in WT,OE and mutant myb13 plants were analyzed,and 1230 and 119 differential genes were obtained among WT-vs-OE and WT-vs-myb13groups,respectively.In order to further study the stress resistance mechanism of tomato under salt stress,the functional annotation of GO and KEGG enrichment analysis were performed on the differential genes,and clustering analysis was performed on the differential genes.The results showed that the knock-out of Sl MYB13 enhanced the transcription of WRKY transcription factor WRKY6.The expression of EDS1 was up-regulated,suggesting that Sl MYB13 might regulate salt tolerance of tomato through an SA-dependent pathway.The expression level of E3 ubiquitin ligase XERICO was decreased,while that of PUB23 was increased,suggesting that Sl MYB13 might be related to ABA biosynthesis.