Studies On The Construction Of Fritillaria Ussuriensis Maxim CAS Gene RNAi Vectors And Establish Genetic Transformation System

Fritillaria ussuriensis maxim is one of the traditional precious Chinese herbs, it has highmedicinal value, can relieve cough and lungs, Fritillaria alkili is one of the main activeingredients of Fritillaria ussuriensis maxim. However, in recent years, due to the continueddestruction of the environment,coupled with Fritillaria ussuriensis maxim growth environmentdemanding,and it has the characteristics of two dormant seasons, when planting itstime-consuming, those are resulting the yield in a sharp decline. In Fritillaria ussuriensismaxim the Fritillaria alkali content is very low, thus leading to a shortage of Fritillaria. Manyscientists at home and abroad are working on clarify the plant secondary metabolitesbiosynthetic pathway, in depth study of the structure and function of the key enzyme genes,aspect to use biological techniques to regulate the activity of these key enzyme genes, toachieve the purpose of improving the purpose product content. cycloartenol synthase wasthought as a plant sterol biosynthesis pathway key enzyme, CAS and DS compete2,3-squalene oxide precursors to make this common metabolic flowing to sterols and triterpenoidstributary. In plant sterol biosynthesis pathway CAS is considered as the first key enzyme.so theactivity of CAS may directly affect the biosynthesis of steroidal alkaloids. RNAi gene silencingtechnology is the recent discovery of a phenomenon triggered by dsRNA, It is mediated by thespecificity dsRNA degradation of homologous mRNA sequences and thus hindered theexpression of the target gene. Therefore, inhibition of CASgene expression, reducingcycloartenol synthase’s activity, will enable the2,3-oxidative squalene metabolic flux isblocked or completely closed, thus further validate its effect on Fritillaria alkili biosynthesis.1. Establishment the Regeneration System of Fritillaria through organogenesis and somaticembryogenesis2.In this study, we use Fritillaria bulbs as material, and use RT-PCR technique successfullyobtained Fritillaria CAS gene fragment786bp.3.According to the obtained gene fragment specific primers were designed to amplify CASgene’s sense and antisense interference fragments, after that connected the sense andantisense interference fragments’s into the expression vector pMHZ111, pMHZ112,then identified by conventional PCR primers and universal primers, indicating interferencevector pMHZ111-CAS and pMHZ112-CAS were constructed successfully.4.After that using vector pMHZ111-CAS and pMHZ112-CAS transformed Agrobacterium EHA105, then using Agrobacterium-mediated transformed Fritillaria bulbs. Laied thefoundation for further elucidation of CAS in Fritillaria alkaloid biosynthetic pathways in thefunction.