Transcriptomic Analysis Of The Endangered Medicinal Plants Atractylodes Lancea(Thunb.)DC.

The Atractylodes lancea(Thunb.)DC.is a kind of perennial herb in Compositae;its rhizome,as a commonly used clinical medicine,has effects of dampness and spleen,dispelling wind eyesight and so on.The A.lancea is one of the geo-authentic crude drugs and recognized as good variety in Jiangsu province,especially in Maoshan area.However,in recent years,the distribution area and population decline became apparent tendency because of various human factors and the biological characteristics of the plant,such as the poor ability of recovering and pollination barriers.At present,the Atractylodes lancea(Thunb.)DC.has been listed as one of the 4 kinds of endangered medicinal plants by Jiangsu province.The main pharmacological components in A.lancea are sesquiterpene,which has a variety of pharmacological activities,and mainly produced by MVA pathway.However,there are few studies on the molecular mechanism of the sesquiterpene formation.In this paper,we constructed the transcriptome database of rhizome and leaf,analyzed the biosynthetic pathway,cloned and analyzed two key enzymes in the biosynthetic pathway,explored the regulating effect of Methyl Jasmonate on physiological and biochemical characteristics of key enzymes and constructed the callus system.The main contents and results are as follows:(1).In this study,the transcriptome platform of rhizome and leaf in A.lancea was established.126072 unigenes were obtained by splicing and assembling.and the 45.56%unigenes were annotated in at least on database.84 unigenes were related with terpenoid skeleton biosynthesis were screened;50 unigenes were associated with the downstream pathways synthesis,in which,9 unigenes,24 unigenes and 17 unigenes were related with monoterpene synthesis,tirterpene and sesquiterpene synthesis and diterpene synthesis,respectively.The differental gene expression results showed that the up-regulated genes in rhizome and leaf were related to their own growth characteristics.The expression of key enzymes related to sesquiterpene biosynthesis in rhizome and leaves have obvious differences.In terpenoid skeleton synthesis pathway,AACT,HMGS,HMGR,PMK,GPS expressed significantly higher in rhizome than in leaf;in the downstream synthesis pathway,the sesquiterpene synthase genes were up-regulated in rihzome while the tieterpene synthase genes were up-regulated in leaf,which means that key enzymes associated with sesquiterpene biosynthesis mainly expressed in rihzome,regulating the production of sesquiterpene metabolites.In addition,we also found 35 candidate P450 involved in modification of terpenoids and 8 candidate transcription factors involved in regulation of sesquiterpene biosynthesis,which is helpful to reveal the regulation mechanism of sesquiterpene biosynthesis in c pathway in A.lancea.(2)The full-length cDNA(2018bp)of Al-ses1 gene was obtained with an open reading frame of 1644 bp,encoding 547 amino acid,which has 72%similarity compared with the amino acid sequence of the sesquiterpene synthase in Matricaria chamomilla var.Recutita.The deduced Al-ses1 protein sequence contained two sesquiterpene synthase conserved motifs,RRX8W and DDXXD,respectively.Phylogenetic analysis showed that Al-ses1 was belonged to the TPSa family,which was a typical sesquiterpene synthase.qRT-PCR results showed that Al-sesl was highest expressed in rhizome,speculating that it may regulate the synthesis of sesquiterpene constituents in rhizome.(3)The full-length cDNA(2149bp)of AlHMGR gene was obtained with an open reading frame of 1752 bp,encoding 547 amino acid.The physical and chemical analysis showed that the molecular weight of the protein encoded by this gene was 62689.4 and the pI value was7.47,which was a hydrophobic protein.Phylogenetic analysis showed that it had a close relationship with HMGRs in compositae,which was a typical HMGR family enzyme.The qRT-PCR results of showed that the expression of the gene in rhizome was higher than that in other tissues.(4)The physiological and biochemical indexes and key enzymes expression were analyzed by stimulating A.lancea.with methyl jasmonate.The results showed that methyl jasmonate can trigger lancea defense system in A.lancea,Increaseing the activity of POD and CAT and inhibiting the activity of SOD;The key enzymes HMGR,AlFPPS,AlHMGR and Alses-1were high expressrd instantly controlled by methyl jasmonate.The results showed that after the treatment with methyl jasmonate,the key enzymes related to defense expressed high due to starting its defense mechanism and then regulated the synthesis of secondary metabolites.(5)On the basis of previous studies,orthogonal experiment was designed to optimize the ratio of growth hormone and we established a stable callus growth system of A.lancea.The results showed that disinfecting 15 min could effectively eliminate the bacteria on the surface of seeds and can obtain the better-growth aseptic seedlings.Howerer,it will decrease the germination rate of seeds.The best hormone ratio of callus formation of the explants was 1.0mg/L2,4-D,0.1mg/L6-BA and 0.2mg/LNAA.the callus induction rate was the highest in this medium.