Study On Expression Of Genes Involved In Swertiamain Biosynthesis In Swertia Mileensis Based On Transcriptome Sequencing

Swertia mileensis is one of traditional chinese herbs which is usually used to treat icteric hepatitis.The main active ingredient of S.mileensis is swertiamarin with good liver protection and cholagogue.The sources of S.mileensis mainly depend on wild resources.On the one hand,the wild resources tend to be exhausted owing to the over-use.On the other hand,the market demand is also becoming more and more difficult to meet.Exploring the genetic basis of the swertiamarin in S.mileensis and finding the key genes involved in the biosynthesis of the swertiamarin,which can lay the foundation for synthesizing swertiamarin in vitro.In this study,the high throughput sequencing by Illumina Hiseq 2500 was used to sequence its transcriptome of S.mileensis.9 candidate genes associated with the biosynthesis of swertiamarin were found,including GPPS-1,GPPS-2,GES,G10H-1,G10H-2,IS,DL7H-1,DL7H-2,SLS.The expression of related genes and the contents of swertiamarin were tested.The correlation was analyzed between them as well.The main research contents were as follows:A total of 685,643 unigenes were assembled with an average length of 901 bp through transcriptome sequencing of S.mileensis.Among them,42,954 unigenes were annotated in the public databases NR,SwissProt,COG,KOG,GO and Pfam.The results showed that 43 unigenes were involved in the production of intermediate,11 unigenes catalyze the synthesis of terpenoids skeleton,and 17 unigenes may participate in terpene post-modification in the three steps of biosynthesis of swertiamain.And 9 candidate genes were found,including GPPS-1,GPPS-2,GES,G10H-1,G10H-2,IS,DL7H-1,DL7H-2,SLS.Quantitative real time PCR technique was used to analyze the expression of candidate genes during different growth periods of S.mileensis.The results showedthat the expression patterns of GPPS-1,GPPS-2,GES,G10H-1,G10H-2,IS,DL7H-1,DL7H-2 were similar.The expressions of these 8 genes were the highest in the fruit period and lowest in seedling stage.However,the expression pattern of SLS was different,which highest expression was in the blooming period and lowest in seedling stage.The content of swertiamarin in S.mileensis was determination by HPLC in different growth periods.The results showed that S.mileensis presented the highest content of swertiamarin in the blooming period.SPSS 19.0 software was performed to analysis the corratins between content of swertiamarin and expression of genes in the different growth periods,The analysis revealed that there were no significant associations between them.Expression of candidate genes and the content of swertiamarin were determinated using the same methods after the treatment of menthyl jasmonate(MeJA).The result showed that the MeJA could improve the expressions of GES,G10H-1,G10H-2,IS,DL7H-1,DL7H-2 and affect the expression of SLS,but no obvious affection on the expressions of GPPS-1 and GPPS-2.The highest content of swertiamarin appeared in the 48 h after the treatment of MeJA.The expressions of G10H-2,DL7H-1 present positive correlations with the content of swertiamarin.The cDNA sequences of DL7H-1 was obtained by RT-PCR technique.DL7H-1gene of full-length 962 bp,with a 948 bp ORF,encoding 315 amino acids.The DL7H-1 in S.mileensis has the most similarity with Vinca minor by NCBI Blast.