Study On Extraction Of Active Component And Cloning Of HMGR Gene Related To Alkaloids Biosynthesis In Fritillaria Unibracteata

F.unibracteata is the dry bulb of manifold plants which belong to the genus Fritillaria of Liliaceae. As one of the most important genuine medicinal materials in Sichuan province, F.unibracteata can eliminate intrinsic heat and wet lung and can be used as expectorant and antitussive, the central effective constituents of which are alkaloids and saponins.Orthogonal experiments was first designed to optimize the extraction technology of total alkaloids and total saponin, and then the total alkaloids and total saponin of 8 different varieties and F.unibracteata origin were investigated respectively based on the optimum extraction method. These results can offer the theoretical gist to industrialized extracting and varietal selective breeding.With aqueous ammonia, ether precipitation total alkaloids extraction from F. unibracteata, orthogonal experimental was designed to inspect extraction time, the ratio of gardenia to liquor, extraction frequency on the three factors on the Extraction Technology, Screening F. unibracteata optimum extraction conditions. With the spectrophotometry method formulation of 8 different varieties, F. unibracteata origin of the total alkaloids content analysis. The best conditions from orthogonal experiments as follows: the extraction time of 36 hours, the ratio of gardenia to liquor of 40, extraction frequency of twice. F. unibracteata total alkaloids content lower than other species, and there is a difference in the same species, different origin Beimu of the total alkaloids content.With methanol extraction, ether precipitation total saponin extraction from F. unibracteata orthogonal experimental designed to inspect the size of material, extraction time, methanol concentration on the three factors on the Extraction Technology, Screening F.cirrhosa optimum extraction conditions; with the alcohol extraction of Gravimetric Method formulation of 8 different varieties, F. unibracteata origin of the total saponin content analysis. Results from the best conditions for the extraction time of eight hours, size of the 100 projects, with the analysis of pure methanol concentration. F. unibracteata total saponin content higher than irbesartan mother, Zhejiang Baimu; The same species, different origin Beimuof the total saponin content there is a difference.The experiment otherwise do the cloning of the key enzyme (HMG-CoA reductase) genic segment from F. unibracteata which is in the biosynthetic public pathway of sterides alkaloids and saponin. The performances of three extracting methods of the Total RNA in bulb of F. unibracteata that contain large amounts of lipin,polysaccharide compounds and phenolic were compared with in this report. The results showed that the modified SDS acid phenol was capable of efficiently removing polysaccharides, and the brightness 28S rRNA of the RNAs isolated by the modified SDS acid phenol was two times as much as that of 18S rRNA, A260nm/A280nm stood at 1.85, A260nm/A230nm stood at 2.06,the RNA productivities from F. unibracteata were 73μg/g. the one and only segment obtained by RT-PCR is 300bp according with deducible length from Primer design. the HMGR genic segment obtained established foundation for Cloning of full length cDNA genic and function and the biosynthetic pathway investigation.