Primary Research On RNAi-induced Silencing Of RsPrxl Gene In Radish

Peroxidase is involved in plant growth and metabolism, especially, anthocyanin metabolism. Inorder to explore the mechanism peroxidase acting on anthocyanins, a construct pART-rsprx1i containingthe partial sense and corresponding antisense sequences was constructed by the Chinese red radishperoxidase gene rsprx1fragments. Agrobacterium strain carrying a T-DNA expressing the ihpRNAtransgene was injected into the growing radish about65days after sowed. Samples used for analysis wereharvested every3days after injection, Agrobacterium containing the pART-27and water wereused as controls. The following main results were obtained:1. AntioxdasePOD：Peroxidase activity of radish was lower consistently in pART-rsprx1i group than controls,and at the lowest (9.82×103U/g·FW) on21th day after injeted (p<0.01). Significant effect of rsprx1geneinhibition occurred in late stage after injection.CAT：Catalase activity is significantly higher in pART-rsprx1i group than the controls on21thday after injection (p<0.01), the increase of CAT activity could be understood as a response to keep freeradical levels result of the lack of peroxidase.H₂O₂：H₂O₂content was enhanced significantly throughout in pART-rsprx1i groupcompared with water group (p<0.01), but only significantly higher than pART-27group on18th day and21th day (p<0.05),Soluble protein：Content of soluble protein decreased from18th day, and was lower than controls,with the lowest content on21th day after injeted (p<0.01). Effect of rsprx1gene inhibition acting onsoluble protein occurred in late stage after injection.2. Anthocyanin：Anthocyanin content was higher in pART-rsprx1i group than controls, the peakswere at9th and12th day (p<0.01). There are difference on content and variety in pART-rsprx1igroup comparing with controls by the analysis of HPLC. 3. RT-PCR：The mRNA level of rsprx1in the pART-rsprx1i group was significantly lower thancontrols on21th day. Expression of anthocyanin biosynthesis genes CHS,CHI,F3H,DFR,LDOX werehighest in pART-rsprx1i group on9th day, which coincided with the expression of TT8and MYB75.Rsprx1from radish was RNAi suppressed, peroxidase can participate in anthocyanin metabolicpathway, and rsprx1gene can affect the biosynthesis of anthocyanin in molecular level and acylatedanthocyanidins. The improved CAT activity was also related to rsprx1gene supression,as well as contentof soluble protein and H₂O₂.