Identification Of Jasmonic Acid Regulatory Gene GH3 Family And Analysis Of Disease Resistance And Wounding In Potato

Jasmonic acid(JA),as an important plant hormone,plays an important regulatory role in the process of plant growth,disease resistance and mechanical damage.Gretchen Hagen 3(GH3)family is an important gene involved in the regulation of jasmonic acid signaling.It mainly converts jasmonic acid into biologically active compounds to induce changes in the expression of response genes.As there are few studies on the jasmonic acid signal pathway genes in potato,there is no report on the function analysis of related GH3 protein.Combining the results of whole potato genome sequencing,using bioinformatics technology to analyze the structure and evolution of potato GH3 protein.Screening of potato response to jasmonic acid and injury stress related candidate genes.Analyze the protein expression location in plant cells,jasmonic acid-induced protein metabolite detection in vitro and the response of transgenic plants to jasmonic acid,bacterial wilt and damage.Finally revealed the molecular mechanism of potato StGH3.1 and StGH3.5 in the regulation of jasmonic acid.Provide theoretical and experimental support for related research.The main results are as follows:(1)In this paper,we firstly identified potato GH3 and other 47 sequenced species(from algae to angiosperms)GH3 proteins using bioinformatics methods,and compared the structural composition and characteristics of all proteins.Differences in binding protein sequences divide GH3 proteins into three subfamilies: Subfamily ? proteins contain conserved serine residues and may be involved in the regulation of jasmonic acid.Subfamily ? proteins contain conserved lysine residues,which are related to auxin regulation.These two subfamily proteins are relatively large,and there are two subfamily proteins in ferns.Therefore,it is inferred that GH3 protein originated from ferns in the plant kingdom.There are few related studies on subfamily ?,and most of the family members are concentrated in the Cruciferae in the pointed plant.Only a few studies have demonstrated that it may be involved in the regulation of salicylic acid.Research on potato GH3 protein showed that only StGH3.1 and StGH3.5 proteins in subfamily I interact with jasmonic acid and may be involved in the regulation of jasmonic acid.The analysis of the changes in gene expression of potato under methyl jasmonate and injury stress showed that the expression of StGH3.1and StGH3.5 genes changed at a higher level under these two stresses.It is further confirmed that StGH3.1 and StGH3.5 genes are involved in the regulation of jasmonic acid.(2)The test results on the subcellular localization of the protein showed that StGH3.1and StGH3.5 proteins in tobacco cells stained by DAPI had superposition signals in the nucleus,while red fluorescence was detected by signals in the cell membrane or cytoplasm.The results of co-localization of Arabidopsis protoplasts combined with cell membrane,cell nucleus,endoplasmic reticulum and Golgi marker showed that only the overlapping signals were detected in the nucleus and endoplasmic reticulum,but no superimposed signal in the cell membrane and Golgi apparatus.Combining the two test results confirmed that StGH3.1and StGH3.5 proteins are mainly expressed in the nucleus and endoplasmic reticulum of plant cells.(3)Prokaryotic expression technology is used to induce the production of StGH3.1 and StGH3.5 active proteins in vitro and react with jasmonic acid.The results of detection of reaction product components by liquid chromatography and mass spectrometry showed that the induced products of StGH3.1 and StGH3.5 mainly contained jasmonic acid isoleucine conjugate(JA-Ile).It was confirmed that StGH3.1 and StGH3.5 proteins can interact with jasmonic acid and produce JA-Ile in vitro.(4)Based on the phenotypic difference of the Arabidopsis mutant Jar1/At GH3.11 under jasmonic acid stress,the potato StGH3.1 and StGH3.5 genes were transferred into the mutant to study the phenotypic difference after jasmonic acid stress,showing that StGH3.1 and StGH3.5 transgenic lines and wild-type Arabidopsis thaliana seed germination and seedling root development were inhibited;while the growth and seed germination of Jar1 mutants were not affected by jasmonic acid.This confirms that the functions of StGH3.1 and StGH3.5 genes are basically the same as those of the Jar1 gene.(5)Construction of StGH3.1 and StGH3.5 gene overexpression and RNAi interference transgenic lines,jasmonic acid-treated transgenic potato tissue culture analysis showed that the transgenic potato under 10 ?M jasmonic acid treatment compared with untreated plants,In addition to the RNAi interference plants of StGH3.1 and StGH3.5,overexpression and wild-type plants promoted the growth of plant roots and main stems;while the plant development induced by 50 ?M JA was significantly inhibited,and only RNAi interfered with the growth of the plant normal.Therefore,it is confirmed that the potato StGH3.1 and StGH3.5 genes are regulated by jasmonic acid.(6)The leaves of each strain were induced to damage,and the content of endogenous JA-Ile in the plant was detected at different induction times.The results showed that the JA-Ile content of the transgenic lines OXStGH3.1 and OXStGH3.5 was significantly higher than that of the transgenic lines OXStGH3.1 and OXStGH3.5 under different stress times.Other strains,and RNAi interference strains have lower hormone content.The results of R.solanacearum infecting potatoes showed that the leaves of RNAi StGH3.1 and RNAi StGH3.5appeared wilting;the colony and leaf peroxide staining of the potato induced by the docking bacteria was studied,and the number of colonies and leaf failure in the RNAi interference strain The accumulation of oxides is higher than other strains.Two experiments finally showed that StGH3.1 and StGH3.5 genes are involved in the stress response of plant disease resistance and damage,and both are related to the regulation of jasmonic acid.(7)The developmental changes of root and stem length of hydroponic potato induced by jasmonic acid indicated that the induction of low concentration of jasmonic acid could promote plant development,while the high concentration of jasmonic acid could inhibit its development.And the deletion of StGH3.1 and StGH3.5 genes could reduce the plant's response to exogenous jasmonic acid on plant development.On the other hand,the statistical results of the growth and development of the transgenic lines showed that the transgenic line with the largest phenotypic difference was the StGH3.5 overexpression line,which may have induced the development of potato lateral branches;but the difference in tuber weight,root length and stem length was not significant.In summary,this study clarified the evolutionary relationship of GH3 protein in the plant kingdom,and the earliest GH3 protein appeared in ferns.According to the difference of amino acid residues in the protein secondary structure,the GH3 protein family is divided into three subfamilies,which are related to jasmonic acid,auxin and salicylic acid signals respectively.Through bioinformatics analysis and analysis of gene expression in mechanical damage and MeJA induction,it is predicted that the potato genes involved in the JA signal pathway that induce JA to synthesize JA-Ile are StGH3.1 and StGH3.5.These two genes are similar in function to the Arabidopsis Jar1 gene.In vitro potato induction and potato disease and injury stress,JA-Ile can be induced to regulate the jasmonic acid signal pathway,which plays an important role in improving plant disease resistance and resistance to mechanical damage.