Application Of Virus-induced Gene Silencing Approach In Metabolic Pathway Analysis Of Camptotheca Acuminata

Camptotheca acuminate Decne is the traditional and precious medicinal plants in China,it is the main source of efficient anti-tumor drug camptothecin. Camptothecin is belong to the monoterpene indole alkaloid, which is the major physiological active substance of Camptotheca acuminata. As the vast majority of medicinal plant, Secondary Metabolite such as camptothecin accumulation is very low in Camptotheca acuminata, which is cann’t meet the market demand. Elucidation of camptothecin biosynthesis pathway is the prerequisite of increase camptothecin accumulation by regulate secondary metabolic. To achieve the cloning and functional characterization of camptothecin biosynthesis-related enzyme genes,complete metabolic pathway analysis, using a fast and effective method to screen and identificate genes which is became very important.VIGS(Virus-induced Gene Silencing) is a method to research gene function, which theory is the virus carrying target gene infects plants, the gene of plants silencing and causing phenotypic variation of plants, is the basis of the target gene function study. VIGS is a technique based on the plant defense RNA virus mechanism, which essence is post-transcriptional gene silencing. Compared with traditional methods of gene functional analysis, VIGS can silence target gene and analyze gene function in infected plant, which can avoid plant transformation, overcome the repeat of gene function and work in different genetic backgrounds, it is a more thorough analysis of gene function. Hence, the VIGS is considered to be a powerful tool for gene functional study in plants, since established has been deeply researched and widely used. Such as tobacco, tomato and other plants, it is used to research functional gene which is involved in disease resistance, growth or metabolic regulation.Camptotheca acuminate Decne is the research object in this thesis. Focus on the use of VIGS technology combined with co-expression analysis, to screen and identify the key enzymes which involve in camptothecin metabolism pathway, such as CaG10 H, CaHGO,CaTDC1 and CaSTR, thus can preliminary analyze camptothecin metabolism pathway. After design the specific primers of genes, we can clone the genes through PCR, then constructTRV-VIGS plasmid gene vectors, and infect leaves of Camptotheca acuminata seedings by Agrobacterium tumefaciens GV3101 which contain TRV-VIGS plasmid. After 3-4 weeks, we can extract RNA and CPT of the VIGS-treated leaves, which are analyzed by qRT-PCR and HPLC. Compared with the empty vector control, the experimental group gene expressions of four genes are decreased by 60%-85%, the experimental group camptothecin and10-hydroxycamptothecin accumulations of four genes are decreased by 35% and 30%around.Finally, we can conclude is feasible of VIGS in plant of Camptotheca acuminata experiment. Hence, we can use VIGS technology combining with co-expression technology to analyze of camptothecin metabolism pathway, screen candidate genes function of cytochrome P450 gene family, to identify genes position and fuction in camptothecin metabolic pathway. The result of experiment is a foundation to research gene function of camptothecin metabolic pathway gene or other pathway gene in Camptotheca acuminata.