| In China, there are various and vast number of medicinal plants which are quite efferctive for clinical treatment. However, most of the medicinal plants had complex genetic background, little transcriptome information and with limited studies on key enzyme genes involved in efficacy component biosynthesis. Defficiency of gene sequences becomes bottlenecks for medicinal plant studies.Combination of express sequence tags (EST) technology and the next generation sequencing technologies provide a good opportunity for obtaining abundant gene sequences and information for medicinal plants. Based on transcriptome dataset of medicinal plants, a magnitude of ESTs were obtained which were used for functional genes exploring and screening in medicinal plants. The transcriptome data of Camptotheca acuminata, Ganoderma lucidum and Panax quinquefolius were applied for further analysis to acquire key enzyme genes of these plants.C. acuminata is a Nyssaceae family plant which is called "happy tree" and indigenous in southern China. C. acuminata produces the terpenoid indole alkaloid camptothecin (CPT) which exhibits clinical obvious effects for various cancers and HIV-virus. This study applies the next-generation454GS FLX Titanium platform on the transcriptome study of happy tree young leaves to discover genes involved in CPT biosynthesis. A total of74,858high-quality ESTs were generated and30,358unique sequences (Unigene) were acquired after GS De Novo software assembly. Based on the further searching of the dataset,20genes (including521ESTs) possibly involved in the upstream pathway were found, of which13genes have not previously reported. Among the13genes, geraniol-10-hydroxylase (G10H), secologanin synthase (SCS) and strictosidine synthase (STR), are the most key ezyme in the pathway which have not been cloned. Candidate genes of the three transcrips were cloned and and analyzed. Expression analysis demonstrated that these genes showed higher expression level in young leaves and petiole as well could be induced by methyl jasmonic acid (MeJA). Genes of cytochrome P450and glycosidase possibly play roles in the branch way of CPT biosynthesis. Based on manual screening and expression profile,7CYP450genes and1glycosidase transcript were selected to be likely participated in the downstream pathway. In addition,21transcripts were discovered to have similarity to the multi-drug resistance genes which could be involved in the transport of secondary metabolism. After tissue expression analysis, three of the21were selected to be candidate genes in CPT transport which had higher level in young leaves than in the old.Ganoderma lucidumia, belonging to Ganoderma genus, Polyporaceace, Basidiomycota, is a medicinal fungi which showed effect on immune system regulation and anti-fatigue. The natural product triterpene ganoderic acid is one of the main pharmacological effect gradients in G. lucidumia. In this study, the454GS FLX Titanium sequencing platform was applied for sequencing of the fruit body transcriptome of G. lucidumia. After454running,479,579ESTs were generated. A total of41,068unigenes were obtained after sequence assembly and22,381unigenes had been annotated against the public databases. Based on BLAST analysis,23sequences was discovered to be likely to participate the lanosterol biosynthesis which was the skeleton of ganoderic acid. A total of171sequences annotated to CYP450s was found from the dataset which probably played roles in the steps after lanosterol biosynthesis. After classification of the CYP450s,112sequences were selected to be as candidate genes for ganoderic acid biosynthesis. G. lucidumia was a type of white rot fungi which can degradate lignin in the environment. In the dataset of fruit body transcriptome, six unigenes were discovered to be involved in the biosynthesis of lignin degradation. All the selected sequences provided abundant resource for further study of biosynthesis of triterpene ganoderic acid and degradation of lignin.P. quinquefolius, a long-lived forest perennial herb, belongs to Panax genus, Nyssaceae family. The natural ginsenosides exhibit excellent clinical effect on the enhancement of immune syntem and tumor treatment. This study searched transcription factor (TF) transcripts which might be involved in regulation of terpene ginsenosides biosynthesis and stress resistant through analysis of high-throughput transcriptome data. Based on the comparison of the four public databases (Nr, Nt, Swissprot, Kegg) and keyword searching,936transcripts were discovered to and annotated to be transcription factors genes. Among these sequences,753unigenes were classified into48plant TF gene families according to the plant TF database. A total of45unigenes, including23contigs and22singltons, had been sorted as WRKY TF transcripts. In this article, after MeJA treatment, a WRKY transcript which was greatly induced by MeJA was screened for cloning and identification which was named as PqWRKY1. Expression of PqWRKY1gene in Arabidopsis showed the expression level of genes in terpene biosynthesis were higher in the transgenic lines. Phynotype analysis indicated that expression of WRKY1gene could enhance salt and drought stress tolerance in transgenic Arabidopsis. This result suggests that PqWRKY1might participate in terpene ginsenosides biosynthesis and stress-resistance in P. quinquefolium.To sum up, the transcriptome of C. acuminata and G. lucidum were sequenced and abundant of ESTs were acquired using high-throughput sequencing platform of the next generation. Many genes likely involved in CPT and GA biosynthesis were obtained using the annotation result. In addition, analysis of TF genes was carried out using the dataset of high-throughput sequencing of P. quinquefolius. A transcript was cloned and identified which probably played roles in triterpene biosynthesis and stress ressistance. This study provides a lot of precious gene resources for the molecular mechanism study of secondary metabolites biosynthesis. Meanwhile, the study lay foundation for natural drug production and medicinal plant molecular breeding. |
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