Medicinal Ingredients Analysis In Different Parts Of Polyphylla And Prokaryatic Expression Of SQS Genes

Paris polyphylla is blindly hemostatic, immunomodulatory and other pharmacological effects of traditional Chinese medicine. P. polyphylla genus has 17 species and 10 variants. The main active ingredient of steroidal saponins, medicinal roots as the main site, but the roots need 3-5 years to be used as medicine, resource regeneration is slow, the aerial parts of P. polyphylla are abandoned. Therefore, selecting high-quality plant species, developing new medicinal resources are important ways to alleviate the shortage of P. polyphylla resources. In this paper, four P. polyphylla roots, stems, leaves, fruits of steroidal saponin and sapogenin are quantitatively detected by HPLC. Establish a new method for the screening of germplasm resources of P. polyphylla, through the P. polyphylla roots and the aerial parts of medicinal ingredients correlation analysis. At the same time, developing new medicinal resources through assessing waste stem, leaf, fruit of medicinal value. In addition, with the technological breakthroughs in tissue culture of P. polyphylla in recent years, make the use of biological engineering technology development possible. Isoprenoid pathway is the biosynthesis pathway of steroidal saponins, but few studies of its key pathway enzymes. This article was cloned cDNA sequence of a key enzyme squalene synthase gene(PpSQS) from P. polyphylla Smith var.chinenisi, and its sequence analysis and prokaryotic expression, for the use of secondary metabolic engineering methods to improve the quality of P. polyphylla, the development of new P. polyphylla resources provides preliminary research foundation. The main work includes the following contents:(1) Establish a effective method of steroidal saponins and saponin extraction and HPLC detection.The content of dioscin, diosgenin and polyphyllin I, II, VI, VII as comprehensive evaluation of P. polyphylla medicinal value. P. polyphylla has highest medicinal value of four P. polyphylla, followed by P. polyphylla Smith var.chinenisi and Paris fargesii, the lowest is P. polyphylla var.stenophylla. Overall, the content of the root of steroidal saponins and sapogenin higher than other parts of P.polyphylla, but polyphylla diosgenin peel contains, polyphyllin I, Hua Paridis leaves contain diosgenin and yams sapogenin, and re-building anther ball leaves contained polyphyllin I levels were higher, which can be used as part of a new ground of choice for the development of medicinal resources. P.polyphylla peels contain higher polyphylla diosgenin and polyphyllin I content, P. polyphylla Smith var. chinensis leaves contain higher dioscin and diosgenin content, P. polyphylla var.stenophylla leaves contain higher polyphyllin I content,which can be used as part of a new choice for the development of medicinal resources. Four P. polyphylla all contain polyphyllin I which content in the roots and in the stems, testas, peels have certain correlation that assess the roots of medicinal value for the use of aerial parts medicinal ingredients possible.(2) Extract total RNA from Paris polyphylla leaves with fresh tissue cultured. According to the reported SQS gene of Paris polyphylla var.yunnanensis(Genebank number: KC442294.1) to design specific primers. The Paris polyphylla Smith var. chinensis SQS gene was amplified by PCR technique, the full-length c DNA of Paris polyphylla Smith SQS was 1296 bp, containing 1227 bp ORF. By Blast analysis, and the similarity of Paris polyphylla Smith var. chinensis with Paris polyphylla var. yunnanensis reached 98%. Verification of the same species of the same gene sequence has high homology.(3) the secondary structure prediction and tertiary model construction reflects P.polyphylla Smith var.chinenisi SQS has more α-helix, the amino acid residues accounted for 64.55 percent of the structure and accounted for 34.96 percent of the cyclic structure, almost no β- fold. Tertiary constrution of P.polyphylla squalene synthase and human squalene synthase have similar activity centers, indicating that P.polyphylla SQS gene and human SQS genes have the same catalytic function. The construction of prokaryotic expression vector of SQS gene which expressed in E. coli E.coli BL21, optimizing P. polyphylla SQS gene the optimal expression conditions in E.coli BL21. Using the method of secondary metabolic engineering, overexpression of the key enzyme gene in the biosynthesis pathway of P.polyphylla Smith var.chinenisi medicinal ingredients, this provide prophase research foundation for improving polyphylla medicinal value.