The triterpenoid saponin content difference is associated with the two type oxidosqualene cyclase gene copy numbers of Pulsatilla chinensis and Pulsatilla cernua

is an important medicinal herb, its dried radix is used to treat the inflammation since ancient China. Triterpenoid saponins are proved to be the main active compounds of genus. The triterpenoid saponin contents vary widely in different species. But no enzyme involved in the triterpenoid saponin biosynthetic pathway was identified in genus. This seriously limits the explanation of the triterpene content difference of species. In this article, we obtained two oxidosqualene cyclase ( ) genes from and by touchdown PCR and anchored PCR. These two OSCs converted 2,3-oxidosqualene into different triterpenoids. The OSC from is a monofunctional enzyme for -amyrin synthesis, while the OSC from is a multifunctional enzyme for lupeol and -amyrin synthesis, and the lupeol is the main product. Then we identified the 260th amino acid residue was the key site for the product difference by gene fusion and site-directed mutant technology. When the 260th amino acid residue was tryptophan (W260) and phenylalanine (F260), the main catalysate was -amyrin and lupeol, respectively. Then we found that the expression of these two genes was strongly correlated with the lupeol-type and -amyrin-type triterpenoid contents in and . Finally, we found the gene copy number difference of these two genotypes leaded to the triterpenoid diversity in and . This study provides useful information for the molecular breeding and quality improvement of and a molecular marker to identify the decoction pieces.