| The active ingredients in the medicinal plant usually belong to secondary metabolites, such as flavonoids, alkaloids, terpenoids. Since the majority of secondary metabolite biosynthesis pathways are complex, and involved a lot of enzymes. In Arabidopsis, some have been described. While, there is a lack of adequate genetic information in the medicinal plants, its secondary metabolic pathways and regulatory mechanisms of secondary metabolites are extreme lack of and thus the development of secondary metabolic engineering is still facing great difficulties. Based on the information provided by structural genomics and the application of high throughput, large-scale analysis, functional genomics is a comprehensive way of study in gene expression, regulation and function, as well as to explore relationship and laws between genes, genes and proteins, and both with the growth and development at the level of genome. Biological research is increased from a single gene or protein to the genome based on functional genomics, while a lot of genes and proteins are systematically studied at the same time. Clarifying the pathways and regulation of secondary metabolie pathway is one of the main contents of functional genomics in medicinal plants. Studies have focused on the transcriptome analysis, medicinal natural product biosynthesis, the key enzyme gene cloning and identification. Ganoderma lucidum and Lonicera japonica are important traditional medicine with high medicinal and economic value, one of the effective compositions of Ganoderma is Ganoderma triterpenoids, one of effective compositions of Lonicera japonica is chlorogenic acid. For these compositions, their biosynthetic pathway is still lack of systematic research. In this study, we make effort to explore the Ganoderma acid and chlorogenic acid biosynthetic pathway in the functional genomics level.Ganoderma, also known as "Lingzhi" is a famous medicinal fungus in China. It is the dry fruiting bodies of G. lucidum and G. japonicrn. It had been proved to possess the pharmacological activities of antitumour, antihypertensive and antiviral. G. lucidum produces a large reservoir of bioactive which mainly included triterpenoids and polysaccharides. It had been reported with the genome size of43.3-Mb and16,113predicted genes. The pathway upstream of the cyclization step includes11enzymes encoded by13genes in G. lucidum. Acetyl-CoA C-acetyltransferases and farnesyl diphosphate synthase are each encoded by two genes in the G. lucidum genome, whereas the remaining nine enzymes are encoded by single-copy genes. A total of219CYP sequences (195functional genes and24pseudogenes) were identified in the G. lucidum genome, and they were classified into42families according to standardized CYP nomenclature. The expression of197CYP genes was investigated using real-time PCR. A total of78genes were found to be upregulated in the transition from mycelia to primordia and then downregulated in the transition from primordia to fruiting bodies. The expression profiles of these genes were highly correlated with that of LAS (correlation coefficient (r)>0.9). Furthermore, their expression profiles are positively correlated with triterpenoid content profiles during development, suggesting that some of these78CYP genes be involved in triterpenoid biosynthesis properly. In addition, there are5P450clusters were co-expressed with LSS.LONICERA JAPONICA FLOS is the dry buds and flowers of Lonicera japonica. Ljaponica is a plant used in traditional Chinese medicine known for its anti-inflammatory, anti-oxidative, anti-carcinogenic, and antiviral pharmacological properties. The major active secondary metabolites of this plant are chlorogenic acid (CGA) and luteoloside. While the biosynthetic pathways of these metabolites are relatively well known, the genetic information available for this species, especially the biosynthetic pathways of its active ingredients, is limited. We obtained one million reads (average length of400bp) in a whole sequence run using a Roche/454GS FLX titanium platform. A total of60036unique sequences were obtained, including21849contigs and38187singletons from flowers of Jizhuahua. A total of54799unique sequences were obtained, inducing14242contigs and40557singletons from leaves of Jizhuahua. And a total of60031unique sequences were obtained, inducing20178contigs and39853singletons from flowers of Damaohua. A total of67954unique sequences were obtained, inducing13201contigs and54753singletons from leaves of Damaohua. All the genes involved in the pathway to biosynthesis of CGA have been found, including putative hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase partial genes. We got8putative HCT/HQT genes full length sequence by RACE.Nearly all of the possible enzymes involved in the biosynthesis of CGA and luteoloside were discovered in L. japonica. Three hydroxycinnamoyl transferase genes, including two hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase genes and one hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) gene featuring high similarity to known genes from other species, were cloned. The HCT gene was discovered for the first time in L. japonica. In addition,188candidate cytochrome P450unigenes and245glycosyltransferase unigenes were found in the expressed sequence tag (EST) dataset. This study provides a high quality EST database for L. japonica by454pyrosequencing. Based on the EST annotation, a set of putative genes involved in CGA and luteoloside biosynthetic pathways were discovered. The database serves as an important source of public information on genetic markers, gene expression, genomics, and functional genomics in L. japonica.Whole genome sequence and EST analysis have a crucial role in the study of medicinal plant secondary metabolic pathways, and provide a wealth of genetic information for the enzyme cloning, identification and speculated the secondary metabolic pathways. In the present study, we analyzed the information about biosynthesis pathway of Ganoderma acid and chlorogenic acid to provide theoretical support for the research of secondary metabolite biosynthesis pathways, and make better development of natural medicinal resources in the future for the purpose of metabolic engineering. |
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