High-throughput Sequencing Of Hypericum Perforatum Transcriptome And Research On Germplasm Resources In Qinling Mountains

Hypericum perforatum L.(St.John's Wort)is a widely known medicinal herb used mostly as a remedy for depression.Germany use it as antidepressants more than 100 years of history.St.John's wort extract and its preparation compared with the traditional chemical antidepressants,it has good efficacy and less side effects.Further research found that H.perforatum also has a good anti-viral,anti-tumor,anti-oxidation,anti-inflammatory,anti-cancer and antibacterial effects.The worldwild demand of H.perforatum is growing,making it become one of the three most popular herbal in the world.Worldwide large-scale development and utilization,led to H.perforatum wild resources depleted,can not longer meet the market demand.Accompanied by the growing demand of H.perforatum and a gradual reduction of its wild resources,to improve the content of the active ingredients and cultivate new varieties with high quality have become the most urgent and key problems in the development of H.perforatum resources.To carry out breeding more efficient,it is necessary to understand the genetic diversity of germplasm resources of H.perforatum.Secondly,study on the key factors of the synthesis and accumulation of active components of H.perforatum and related genes,can provide scientific basis for the breeding and quality control.In view of the above-mentioned facts,based on samples collected from Qinling Mountains(China),we studied genetic diversity of H.perforatum from three aspects,morphology,content of effective components and DNA levels.We employed Illumina HiSeq 2000 to evaluate the transcriptome of H.perforatum throughout its life cycle.Arabidopsis transcription factor,AtPAPl,were transferred into H.perforatum.The content of active ingredient significantly increased in transgenic lines.Provide information of studied of H.perforatum secondary metabolites biosynthesis.The main results and conclusions are as follows:1.Collected the germplasms of H.perforatum more than 200 copies from Qinling Mountains,and detailed description of their morphological characteristics.The genetic diversity among 26 representative germplasms of H.perforatum were studied using SRAP molecular markers and 12 SRAP primer combinations.A total of 183 bands and 153 polymorphic bands were obtained that accounted for 83.6%of the polymorphism.We employed HPLC to determine content of five phenolic components.Analysis by the UPGMA method grouped the germplasms into three main clusters,with coefficients of similarity ranging from 0.57 to 0.97.With similar traits the germplasms clustered into a cluster.Germplasms in Cluster ? were the highest,and contained high content of chlorogenic acid,hyperoside and quercitroside but low content of rutin.Germplasms in Cluster ? were the shortest,and contained few numbers of the black glands on stems.Germplasms in Cluster ? contained most numbers of the black glands on stems.Correlation analysis between morphological characteristics and active ingredient content revealed that BDSS significantly and positively correlated with BDVS(P<0.001)and LBD(P<0.05).While,the hyperoside concentration was significantly and positively correlated with BDVS.Show that the more of black glands on vertical edges of stem,the more of black glands on the leaves,and higher content of hyperoside.2.We employed Illumina HiSeq 2000 to evaluate the transcriptome of H.perforatum throughout its life cycle.We obtained 59,184 unigenes covering the entire life cycle of these plants(SRA050246.2).In all,40,813 unigenes(68.86%)were annotated and 2,359 were assigned to secondary metabolic pathways.Among them,371 unigenes are involved in the production of hypericin,hyperforin,melatonin and flavonoid.Another 2,291(3.87%)unigenes,which have a unique amino acid sequence homologous to type ? polyketide synthase,are classified as potential.,type ?polyketide synthase.Another 2,291 unigenes are classified as potential.Ttype ?polyketide synthase.Our Blastx search against the AGRIS database reveals 1,772 unigenes that are homologous to 47 known Arabidopsis transcription factor families.Further analysis by using MicroSAtellite shows that 10.61%(6,277)of these unigenes contain 7,643 SSRs.18%(1,130)of the unigenes contain multiple SSRs.Real-time quantitative PCR were used to detect the expression of part of the newly obtained transcript from different organs.The expression of genes related to melatonin synthesis were highly expressed in the organs contained chloroplast,consistent with the results of previous studies.While,PKS were highly expressed in the flowers,consistent with the results that the content of polyketide meet the maximum content at flowering phase.3.The Arabidopsis PAP1 transcription factor was transferred into H.perforatum,and 19 transgenic lines were obtained by using Agrobacterium tumefaciens-mediated genetic transformation for the first time.AtPAPl highly expressed in four transgenic line were detected by real-time PCR.Measured by HPLC the transgenic line of H.perforatum had high content of rutin,hyperoside and quercitroside.Line PAP 1-22 had the highest content of rutin,hyperoside and quercitrin.It was 1.94,3.11 and 2.43 times comparing with wild-type,respectively.And it was 1.91,2.97 and 1.99 times comparing with transgenic lines with empty vector,respectively.Total flavonoid content of PAP 1-16 was the highest,1.5 times than the wild type.Real-time PCR were used to detect genes expression which involved in the flavonoid biosynthetic pathway.HpPAL?HpC4H?Hp4CL and HpCHS were more or less up regulated in transgenic lines.Six genes highly expressed in line PAP1-16,such as HpPAL?HpC4H?Hp4CL?HpCHS?HpF3'H and HpFLS.It was 1.51-3.84 times comparing with wild-type.Thus,we hypothesis that the expression of AtPAPl up-regulated the expression of six genes related to flavonoids biosynthesis lead to highly biosynthesis and accumulation of flavonoids.