Mining And Identification Of Genes Involved In Dendrobium Alkaloid And Ginsenoside Biosynthesis Based On Transcriptome Analysis

Medicinal plants is a major source of traditional Chinese medicine, while the secondary metabolites are an important source of new drugs, drug leads and new chemical entities. With the extensive application of medicinal plants in drug development, the demand for medicinal plants is increasing. Long-term disorderly development of medicinal plants is not only caused by the scarcity of resources, but also seriously damage to the ecological environment. The number of endangered species of wild medicinal plants also increased rapidly. The use of biotechnology method to synthesize large-scale medicinal components has become important way to explore drug development. Based on the high throughput sequencing technology, the studies of transcriptome can provide important genetic resources for the medicinal natural product biosynthesis, combined with key gene cloning and identification, as well as large-scale production of medicinal components.In this study, we used the454high-throughput sequencing technology to analyze the transcriptome of Dendrobium officinale and Panax ginseng and discover key genes related to the Dendrobium alkaloids and ginsenoside biosynthesis. Moreover, several genes encoding key enzymes had been cloned and heterologously expressed in E.coli. The content of this study includes the following aspects:1) Dendrobium officinale Kimura et Migo(Orchidaceae) is a traditional Chinese medicinal plant. The stem contains an alkaloid that is the primary bioactive component. However, the details of alkaloid biosynthesis have not been effectively explored because of the limited number of expressed sequence tags (ESTs) available in GenBank. In this study, we analyzed RNA isolated from the stem of D. officinale using a single half-run on the Roche454GS FLX Titanium platform to generate553,084ESTs with an average length of417bases. The ESTs were assembled into38,951unique putative transcripts. A total of69.97%of the unique sequences were annotated, and a detailed view of alkaloid biosynthesis was obtained. Functional assignment based on Kyoto Encyclopedia of Genes and Genomes (KEGG) terms revealed69unique sequences representing25genes involved in alkaloid backbone biosynthesis. A series of qRT-PCR experiments confirmed that the expression levels of5key enzyme-encoding genes involved in alkaloid biosynthesis are greater in the leaves of D. officinale than in the stems. Cytochrome P450s, aminotransferases, methyltransferases, multidrug resistance protein (MDR) transporters and transcription factors were screened for possible involvement in alkaloid biosynthesis. Furthermore, a total of1,061simple sequence repeat motifs (SSR) were detected from36,407unigenes. Dinucleotide repeats were the most abundant repeat type. Of these,179genes were associated with a metabolic pathway in KEGG.2) Panax ginseng C A. Meyer is one of the most widely used medicinal plants. Two454pyrosequencing runs generated a total of2,423,076reads from P. ginseng roots, stems, leaves and flowers. The high-quality reads from each of the tissues were independently assembled into separate and shared contigs. In the separately assembled database,45,849,6,172,4,041and3,273unigenes were only found in the roots, stems, leaves and flowers database, respectively, In the jointly assembled database,178,145unigenes were observed, including86,609contigs and91,536singletons. Among the178,145unigenes,105,522were identified for the first time, of which65.6%were identified in the stem, leaf or flower cDNA libraries of P. ginseng. After annotation, we discovered223unigenes involved in ginsenoside backbone biosynthesis. Additionally, a total of326potential cytochrome P450and129potential UDP-glycosyltransferase sequences were predicted based on the annotation results, some of which may encode enzymes responsible for ginsenoside backbone modification. A BLAST search of the obtained high-quality reads identified14potential microRNAs in P. ginseng. A total of13,044simple sequence repeats were identified from the178,145unigenes.3) Panax notoginseng (Burk.) FH Chen is a perennial herb in Ginseng genus. The precursor substances of ginsenoside are biosynthesized through the mevalonic acid pathway. In this study, we found many genes related to ginsenoside biosynthesis based on the research of the P. notoginseng transcriptome data. The full-length cDNA sequences of PnMVKl, PnUGT02086, and PnUGT13895genes were obtained by RT-PCR strategies. The physical and chemical properties, secondary structure and three-dimensional structure of these proteins were predicted and analyzed, including of the structure and putative functions. Pn MVK1was more abundant in P. notoginseng root than other organisms. PnUGT02086and PnUGT13895were ligated into pET28a vector and transformed into BL21. The proteins of PnUGT02086and PnUGT13895were purified from E.coli, which laid the foundation for the further verification of the function for these genes.