| Salvia miltiorrhiza Bunge(Labiatae)is a familiar traditional Chinese herbal medicine(TCM).Its dry roots or rhizomes are mostly used for treatment of cardiovascular diseases.Because of the advantage in Chinese materia medica and Molecular Biology,S.miltiorrhiza has been paid more and more attention by domestic and foreign scholars,and is described as a medical model plant.Studies on the biosynthesis pathway of active pharmaceutical ingredients of phenolic acids and tanshinones as well as the accumulation mechanism of secondary metabolites in S.miltiorrhiza have also been widely concerned.With the advent of the era of big data,transcriptome,genome and metabolome,which are manipulated by high-throughput sequencing,are increasingly favored by biological researchers.Though analysis of S,miltiorrhiza omics,researchers could obtain more factors that affect S.miltiorrhiza secondary metabolism such as key enzyme genes,important regulatory factors,sensitive inducers,gene families and so on.SABATH methyltransferase gene family is a group of methyltransferase that could methylate of small molecules such as jasmonic acid,salicylic acid and so on.Due to the importance of catalytic substrates and catalytic products,the family members of SABATH methyltransferase gene family are paid more attention.In addition,many studies have shown that methyl jasmonate plays an important role in regulating plant growth,development and plant secondary metabolism.Furthermore,it also could enhance plant defense.While,Jasmonic acid carboxyl methyltransferase catalyzes and regulates methyl jasmonate in plants.Therefore,in this study,we proposed to screen out the Jasmonic acid carboxyl methyltransferase gene by analyzing the phyletic evolution of SABATH gene family in S.miltiorrhiza,and attempt to modulate the methyl jasmonate metabolism in S.miltiorrhiza by regulating the Jasmonic acid carboxyl methyltransferase of S.miltiorrhizo,and to investigate the effects of endogenous methyl jasmonate on secondary metabolism,defense,and development of S.miltiorrhiza.The main results and conclusions are as follows:1.Based on the S.miltiorrhiza genome database,30 members of the SABATH family were screened out and identified.Phylogenetic analyses showed that SmSABATH genes could classify into three subgroups,and 11 pairs of paralogous genes were found.Positive selection analyses using site models and branch-site models indicated that SmSABATH genes had undergone purifying selection for adaptive evolution.Functional divergence analysis suggested that the SmSABATH subgroup genes were divergent in terms of evolutionary rate between groups.Analysis of gene expression patterns found that the SABATH gene family in S.miltiorrhiza responded to methyl jasmonate and mechanical injury.Tissue-specific expression showed that the SABATH gene family in S.miltiorrhiza was primarily expressed in stems and leaves.2.Through analysis the orthologs genes of SmSABATH,and blasting the protein sequences of SABATH gene family in S.miltiorrhiza,a orthologs gene of A.thaliana Jasmonic acid carboxyl methyltransferase with characteristic functional motif of jasmonic acid methyl transferase was screened out in SmSABATH.The cDNA of the gene was obtained by PCR.The length is 1,167 bp,coding 389 amino acids.The sequence comparison showed that this protein is highly similar to the jasmonic acid methyl transferase of S.indicum and E.guttata which belong to the Lamiales.Furthermore,this protein has a characteristic functional motif of jasmonic acid methyltransferase and a typical s-adenyl-L-methionine binding domain.Thus,this protein was named Jasmonic acid methyltransferase of S.miltiorrhiza.The number of Genbank is MH136806.Bioinformatics analysis showed that the protein was non-transmembrane,non-secretory,without obvious hydrophobic and hydrophilic properties.Phylogenetic analysis showed that the cluster relationship of JMT proteins from different species is consistent with the traditional taxonomy.Analysis of gene expression patterns found that the SmJMT is primarily expressed in stems and leaves,and responded to methyl jasmonate and mechanical injury.3.By means of Gateway technology,the overexpression and interference JMT vectors of S,miltiorrhiza were constructed,respectively.An Agrobacterium-mediated gene transfer method was performed to generate transgenic plants with leaf explants from sterile S.miltiorrhiza.Then,the constructed vector was successfully inserted into the genome of S.miltiorrhiza.The positive lines were tested and screened at DNA and RNA levels,respectively.Finally,we gained three high expression lines of Line7,Line9 and Line 10 in overexpression lines,and three lower expression lines of Line7,Linel land Line 12 in interference lines.4.Root scanner was used to analyze the root growth of overexpression and interference SmJMT lines.The statistical results showed that,in overexpression SmJMT lines,root occurrence was earlier than the control,the mean length of root was significantly longer than the control,the projected area,superficial area and volume of root was significantly bigger than the control.Furthermore,the number of root tip was more than the control.While,compared with the control,the root occurrence was later,the mean length of root was shorter,the projected area,superficial area and volume of root was smaller,and the number of root tip was less in interference SmJMT line.In addition,with the method of observing the damaged area of S.miltiorrhiza leaves chewed by 3-age cotton bollworm,the insect tolerance of overexpression and interference SmJMT lines was tested.The results showed that the leaf damage index of the overexpression SmJMT lines was 50%of the control lines.While,the leaf damage index of interference SmJMT lines was twice as high as that of the control lines.The results indicated that the tolerance to cotton bollworm be significantly enhanced in overexpression SmJMT lines compared with the control,while the tolerance be significantly declined in interference SmJMT Vines compared to the control.5.Using ELISA,HPLC and LC/MS detection methords and instruments,the content of jasmonates and mainly secondary metabolites of overexpression and interference SmJMT lines were tested.ELISA test revealed that the content of endogenous methyl jasmonate was significantly elevated in overexpression SmJMT lines compared to the control,while the contents were significantly decreased in interference SmJMT lines compared to the control.In additional,secondary metabolites detection showed that the contents of salvianolic acid B,Tanshinone II A,cryptotanshinone,the total phenolics and total flavonoids in transplanted 2-month-old of overexpression SmJMT lines were significantly increased compared with the control lines.The OEJ-10 lines showed a 1.53-fold,1.59-fold,3.25-fold,1.85-fold and 2.19-fold increase in salvianolic acid B,Tanshinone II A,cryptotanshinone,the total phenolics and total flavonoids compared with the control,respectively.While,the contents of mainly secondary metabolites in transplanted 2-month-old of interference Sm.JMT lines were significantly decreased compared with the control lines.The contents of salvianolic acid,Tanshinone II A,cryptotanshinone,the total phenolics and total flavonoids in iJMT-12 line were 72.2%,55.6%,60.0%,66.2%和 67.6%of the control lines,respectively.6.With the qRT-PCR technology,the expression of genes encoding key enzymes involved in the biosynthesis pathway of methyl jasmonate,phenolic acids and tanshinone were analyzed.The results showed that the expression levels of genes encoding key enzyme of SmLOX,SmAOS and SmJMT in the overexpression SmJMT lines were significantly up-regulated than the control,while those in the interference SmJMT lines were significantly down-regulated compared with the control lines.The genes encoding key enzymes such as SmPAL,SmC4H,Sm4CL,SmTAT,SmRAS and SmCYP98AI4 in the phenolic acids biosynthesis pathway were significantly up-regulated in overexpression JMT lines compared with the control,while those in interference SmJMT lines were significantly down-regulated than the control lines.In additional,the expression levels of genes encoding key enzymes of SmDXS,SmHMGR,SmFPPS,SmGGPPS,SmCPS and SmKSL involved in the tanshinone biosynthesis pathway in overexpressed SmJMT lines were significantly up-regulated than control lines,while those in interference SmJMT lines were significantly down-regulated compared with the control lines.The results showed that the key enzyme genes involved in the pathway of methyl jasmonate,phenolic acids and tanshinone could regulated by the SmJMT gene.7.High-throughput sequencing analysis of overexpression and interference JMT lines and their controls was performed with the method of comparative transcriptome.A total of 30,052 uigenes were obtained.There were 2,052 DEGs were screened out in overexpression samples(998 up-regulated while 1,054 down-regulated).In addition,3,864 DEGs were screened out in interference samples(2,074 up-regulated while 1,790 down-regulated).GO differences enricluhment showed that overexpression and interference samples GEGs were significantly enrichment in cell metabolism,response to stimulation,and cell regulation,suggesting that JMT gene may be closely related to metabolism and defense in S.miltiorrhiza.KEGG pathway differential enrichment analysis showed that overexpression and interference samples were significantly eenrichment in the biosynthesis pathways of phenylalanine and terpenoid,as well as the metabolic pathways of a-linolenic acid,indicating that the JMT gene may participate in regulating the biosynthesis pathway of methyl jasmonate,phenolic acids and tanshinone.The differential expression analysis of the uigenes encoding key enzyme invoved in the biosynthesis pathway of methyl jasmonate,salvianolic acids and tanshinone,showed that the expression level of most uigenes encoding key enzyme in overexpression SmJMT samples were higher than that of control and interference SmJMT samples,while the expression level of most uigene encoding key enzyme in interference JMT samples was lower than that of control and overexpression SmJMT samples.The results of transcriptome analysis of each sample also confirmed the expression difference analysis of real-time quantitative of genes encoding key enzyme.In conclusion,in this study,based on the Smiltiorrhiza genome database,the SABATH gene family was identified in S.miltiorrhiza.Firstly,the phylogenetic evolution analysis,selection pressure analysis and functional divergence analysis of the SABATH gene family in S.miltiorrhiza were performed.Then,the jasmonic acid methyltransferase of S.miltiorrhiza was screened out through phylogenetic evolution analysis,orthologs genes analysis and sequence comparison analysis of the SmSABATH gene family.And then,the bioinformatics and subcellular localization analysis of SmJMT were carried out.Thirdly,using biotechnological means,the overexpression and interference SmJMT transgenic lines were obtained,and the content of jasmonates and mainly secondary metabolites as well as insect tolerance of overexpression and interference SmJMT lines were compared and tested.Finally,DEGs of transgenic and control lines were comparatively analyzed by transcriptome.This study provides a basis for further understanding the function of methyl jasmonate transferase in medicinal plants. |
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