Establishing RNA Interference As A Reverse-Genetic Approach For Gene Functional Analysis In Papaya Protoplast And Its Application

With the whole-genome sequencing of papaya was completed, to develop a platform of convenient, efficient and rapid identification for gene functional analysis is of great significant, while the platform of RNAi-based analyses in protoplasts has those advantages, In this study, we constructed transient expression vector of GFP and the corresponding ihpRNA vector for co-transformation of papaya protoplasts. Our transient RNAi experiments provided information about highly efficient protoplast transformation system:the concentration of the protoplasts(1×106/mL), the vitality of the protoplasts dose(≥90%), plasmid concentration(20μg). The observation by fluorescence microscopy and RT-PCR analysis showed that high RNA silencing efficiency of90%for model target genes such as GFP have been obtained.An analysis of the candidate genes in benzyl glucosinolate (BG) biosynthesis in papaya by the established system was done. We then cloned the corresponding six genes(CYP79A2.1, CYP79A2.2, CYP83B, C-S, UDP-T and ST5a) in BG biosynthesis in papaya though the reference genes in Arabidopsis. After bioinformatic analysis, we constructed the ihpRNA vector of the genes using the method of ligation-independent cloning(LIC) for transformation of papaya protoplasts. Based on RT-PCR analysis, we acquired the ihpRNA vector of best RNA silencing efficiency. Then, we attempted to detect the corresponding changes in the metabolite profile by HPLC. The results showed that the BG content in the protoplasts were decreased by varying degrees. It is indicated that the candidate genes are the genes in BG biosynthesis in papaya. And the BG was not detected in protoplasts transferred by ihpRNA vector pRNAi-CYP83B、 pRNAi-UDP-T or pRNAi-ST5a.This results indicated that protein CYP83B, UDP-T and ST5a, were key regulatory enzymes in BG biosynthesis in papaya.