Evaluation Of Germplasm Resources Quality Based On RNA Sequencing In Atractylodes Lancea

Atractylodes lancea(Thunb.)DC.(Compositae)is a perennial herb and widely prescribed traditional Chinese herb.It is one of the origin plants of Atractylodes and mainly produced in Hubei,Jiangsu,Henan and other provincese.Due to the increasing demand of A.lancea,the wild resources of Maoshan,a Traditional authentic region,have been severely damaged.Now,A.lancea on the market mainly from cultivars of Hubei Yingshan.In this paper,we studied different germplasm resources quality of A.lancea in two regions based on RNA sequencing,Cloned and analyzed the key genes of terbioid biosynthesis.The main research contents are as follows.1.Evaluation of germplasm resources quality based on RNA sequencing in A.lancea.The Illumina Hi Seq2000 platform was used for sequencing of 10 samples of rhizomes and leaves of A.lancea of two origins.Sequencing results showed that the clean reads were: leaves of A.lancea in Maoshan(JL)118,397,448,leaves of A.lancea in Yingshan(YL)91,761,748,roots of A.lancea in Maoshan(JR)152,688,850,roots of A.lancea in Yingshan(YR)110,478,126.All clean reads were assembled into 125,711 unigenes,73785(58.69 %)of which are homologous with sequences in public protein databases(Blastx,Blastp,Swiss-Prot,Tr EMBL,and KEGG),34406(27.37%)of which are classified under one or more GO secondary functional categories,and 9700(7.72%)of which are mapped to the metabolic pathway of KEGG database.Analysis of the differently expressed genes(DEG)of A.lancea in two areas showed: there are 4087 DEG in JL compared with YL,and 2118 DEG in JR compared with YR.In KEGG analysis results,there are three metabolic pathways closely related to primary metabolism: ko00500(Starch and sucrose metabolism),ko00010(Glycolysis)and ko00710(Carbon fixation in photosynthetic organisms)correspond to 40,19 and 18 gene,respectively.At the same time,two metabolic pathways ko00900(Terpenoid backbone biosynthesis)and ko00909(Sesquiterpenoid and triterpenoid biosynthesis)related to the synthesis of terpenoid secondary metabolites were found respectively corresponding to 7 and 4 gene.These data offer an overview of the different transcriptome profiling of A.lancea in two regions.Therefore,this paper provide new ways to study the quality of Chinese herbal medicines and paves the foundation for the follow-up study of terpene synthetase genes in A.lancea.2.Cloning and expression analysis of sesquiterpene synthase gene(Al TPS1)in A.lancea.Based on the above transcriptome data,we found a fragment of the sesquiterpenoid gene(Al TPS1),then amplified sequence of 3 'end and 5' end by RACE technology and obtained the full-length sequence of Al TPS1.The full-length c DNA of Al TPS1 is 1644 bp and encode 547 amino acids.The RT-q PCR method was used to detect the expression of Al TPS1 in different tissues of A.lancea.The results showed that the expression level was higher in flowers and leaves,lower in rhizome and no expression in roots.The recombinant expression vector Pet28a-Al TPS1 was constructed successfully.The Al TPS1 was induced to express by IPTG,and the size of the expressed protein was 64 KD.It is the first time to report sesquiterpenoid in A.lancea and its bioinformatics analysis,relative expression level and protein expression.The results will provide a groundwork for studying sesquiterpenoids biosynthesis of A.lancea.3.Cloning and analysis of DXS gene in A.lancea.Based on the above transcriptome data,we screened a 1-deoxy-D-xylulose 5-phosphate synthase(DXS)gene,a key gene of the terpenoid skeleton synthesis pathway.The c DNA sequence of DXS gene was cloned and the full-length of DXS gene was 2151 bp,encoding 716 amino acids,which has been deposited in Gen Bank with accession number: KY659208.The phylogenetic analysis of the amino acid sequence of the DXS gene showed that the sequence has high homology with the DXS gene of Compositae plants,such as Stevia rebaudiana.The expression of DXS gene in different tissues of A.lancea was detected by q PCR.The results showed that the DXS gene was mostly expressed in leaves,followed by flowers,rhizome and roots.This chapter mainly report DXS gene in A.lancea and its bioinformatics analysis and relative expression level.The results will provide a groundwork for studying the function of DXS in terpenoid biosynthesis of A.lancea.