The Molecular Mechanism Of Tomato SlMYB86 In Response To Nitrate Stress

Secondary salinization is one of the main problems in soil of greenhouse in China.Previous studies showed that the salt composition of these soil is mainly NO₃^-and Ca²⁺.Excess NO₃^-inhibit plant growth and development,which seriously restricts the development of protected vegetables.MYB transcription factors,as one of the largest transcription factors in plants,play important roles in plant abiotic stress.Nitric oxide（NO）is an important signal molecule in plant growth and development and in response to abiotic stress.S-nitrosylation is the main way for NO to exert its biological activity.Thiredoxin（Trx）is the main enzyme regulating the redox state of plants.Trx also functions as the denitrosylase in plants.In this paper,the molecular mechanism of tomato R2R3-MYB transcription factor SlMYB86 in response to nitrate stress was studied.The results are as follows:1.Bioinformatics analysis of SlMYB86 showed that SlMYB86 was closely related to SlMYB26,and the N-terminal of SlMYB86 transcription factor contained two Myb＿DNA-bindings domain belongs to R2R3-MYB type MYB transcription factor.Subcellular localization showed that SlMYB86 was located in the nucleus.After nitrate stress and exogenous application of NO donor sodium nitroprusside（SNP）,it was found that the expression of SlMYB86 increased significantly in tomato seedlings.Biotin transformation experiment showed that SlMYB86 could undergo S-nitrosylation.The recombinant protein of tomato SlMYB86 was induced and purified by prokaryotic expression system,and the antibody was obtained by immunizing mice.Biotin transformation experiments showed that SlMYB86 could undergo S-nitrosylation in vitro.2.Through the analysis of SlTrx promoter on plant CARE website,it was found that SlTrx promoter contained MYB binding site.Luciferase test showed that SlMYB86 activated the expression of SlTrx promoter,.Electrophoretic mobility shift assay（EMSA）further verified the combination of SlMYB86 in the 5’-GTCAAC-3’region.Ch IP-q PCR results showed that SlMYB86 combined with MYB element in C3region of SlTrx promoter.93 proteins that may interact with SlMYB86 were screened by yeast library,and the interaction between E2 ubiquitin binding enzyme and V-ATPase and SlMYB86 protein was preliminarily verified by yeast two hybrid.3.SlMYB86 overexpression transgenic tomato plants were obtained were analyzed their nitrate stress tolerance.The results showed that under nitrate stress,the growth of SlMYB86 overexpressing plants was better than that of wild type（WT）,and the contents of ROS,H₂O₂and O₂^-in SlMYB86 overexpressing plants were significantly lower than that of WT,indicating that SlMYB86 overexpressing plants suffered less oxidative damage under nitrate stress.The activities and expression of antioxidant enzymes of SOD,CAT,POD and APX in SlMYB86 overexpressing plants were higher than those in WT plants under nitrate stress,and the content of SNO,NR activity and S-nitrosolating levels were also higher than those in WT plants.The results showed that SlMYB86 overexpression enhanced the tolerance to nitrate stress.4.The transgenic tomato plants of SlTrx RNAi was obtained,and the tolerance of SlTrx RNAi tomato to excess nitrate stress was analyzed.Under nitrate stress,the plant height and fresh weight of RNAi plants were lower than those of WT plants,and the contents of H₂O₂and O₂^-were significantly higher than those of WT plants.The activities and expression of antioxidant enzymes SOD,CAT,POD and APX in SlTrx RNAi plants were higher than those in WT plants under nitrate stress,and the content of SNO and NR activity were also higher than those in WT plants.The results showed that SlTrx RNAi reduced the resistance of tomato under nitrate stress.