Establishment Of CDNA Microarray Of Salvia Militiorrhiza And Gene Expression Profiling Of Hairy Root

Salvia miltiorrhiza Bunge. as a well-known Chinese herbal medicine has widely used in the treatment of cardio-cerebral diseases. It mainly contains liposoluble phenanthrofurane quinone derivatives and water-soluble phenolic acids. The phenanthrofurane quinone derivatives such as tanshinones have significant pharmacological effects including antioxidation effect, the prevention of inflammation and so on. But there are many diffdrences in origin of Salvia miltiorrhiza, it is important to find the factors which impact its quality and explore the gene of key enzymes in the metabolism pathway. Gene chip is a powerful tool to find new genes. In this paper, we develop the first cDNA chip of Salvia miltiorrhiza and monitor gene expression profiling in the different stage of hairy root, obtain many usefulgenes. The main results are as follows:1. The cDNA library was constructed by stratagene ZAP Express Predigested Vector Kit andZAP Express Pridigested Gigapack Cloning Kits in Invitrogen Company. It contains about 3 × 10~5 clones and insertion fragment is 500~2500bp.2. Totally 4354 genes were singled out from the libraries for cDNA Microarray manufacture.It is the first cDNA Microarray about traditional Chinese herbs.3. 30d, 45d and 60d hairy roots were analyzed chief bioactive ingredients content, growth rateand hybridized simultaneously, total obtained 114 different expressed genes, among which 52 is first find in Salvia miltiorrhiza.4. Based upon BLASTX and BLASTN search similarity in NCBI, 105 genes were noted.5. Based on the gene ontology, total 66 genes were classified into cellular component ontology, molecular function ontology and biological process ontology.6. Based on the KEGG pathway classification. Total 27 genes, which represent 29 metabolic-related enzymes, were analyzed and located in metabolic maps. The genes involved in Carbohydrate Metabolism, secondary Metabolism and Amino Acid Metabolism showed much abundant expression level.7. Northern Blot was used to test the result of cDNA microarray. Two results are identical.8. One Diterpene synthase cDNA fragment copalyl diphosphate synthase ( CPS ) was cloned for the first time. The length is 1479bp, coding 418amino acids, contain Terpene_cyclase_ plant C1 doman.9. Eight P450 family genes were cloned;four were cloned for the first time.10. Eight genes of full length cDNA were cloned for the first time. Including two Aldo-keto reductase, two metallothionein and dienelactone hydrolase, translationally controlled tumor protein, germin-like proteins and ethylene responsive element binding protein.In summary, the first cDNA microarray was established with traditional Chinese herbs, 52 Unigenes were first reported in Salvia miltiorrhiza. One Diterpene synthase, four P450 family cDNA fragments and eight full-length genes were cloned for the first time. The research established a new model to monitor gene expression patterns in Salvia miltiorrhiza, making the clone of key enzyme genes more easily.