Determination Of Important Alkaloids & Analysis Of Relative Gene Expression In Catharanthus Roseus

Alkaloids are nitrogen-containing basic compounds in plants and constitute one of the largest groups of natural secondary metabolites. People have been of great interest in alkaloids because of their pharmacological activities. Catharanthus roseus (C.roseus) contains important anti-tumor terpenoid indole alkaloids (TIAs) such as vinblastine and vincristine. Most of these TIAs are produced at low levels in the natural plants and are difficult to be chemically synthesized due to their complex structures. There remains a need to increase alkaloids production rates for commercial exploitation. In recent years, progress on C.roseus'TIAs'metabolic pathway provides theoretical basis for improving C.roseus'TIAs'production through plant genetic engineering. In genetic engineering, it requires the establishment of rapid approaches for measurements of C.roseus'TIAs. Analytical works on the relationships between the upstream and downstream metabolites'content, and the relationship between the important genes'expression levels and metabolites'contents are also needed.The contents of vindoline, catharanthine and vinblastine in the leave of 25 C.roseus varieties were measured by HPLC. These data provided bases for the choice of proper varieties for future genetic engineering. RT-PCR method was employed to study the expression levels of 4 important genes (tdc,g10h,str,dat) in the metabolic pathway. The relationships between genes'expression levels and TIAs'contents were analyzed.In this study, the following progresses have been made:1. The RP-HPLC condition for determination of vindoline, catharanthine and vinblastine in C.roseus leave extract were optimized. Thress TIAs can be measured in one time.2. The 3 TIAs'contents in the leave of 25 C.roseus varieties were measured. The relationship between vinblastine and vindoline's contents and the relationship between vinblastine and catharantine's contents were analyzed. The result shows vinblastine and vindoline's content are better related.3. The expression levels of 4 important genes (tdc,g10h,str,dat) in 6 C.roseus varieties were studied through RT-PCR. The relationship between gene expression level and TIAs'content were analyzed. The result shows that the expression levels of g10h and str reflect TIAs'contents better.