Research On Apple Polyphenol Oxidase Double-stranded RNA (dsRNAi)

RNA interference is a form of post-transcriptional gene silencing (PTGS) mediated by double-stranded RNA (dsRNA). It is a new developed gene-silencing technique used in studying gene expression regulation and function.Antisense and sense gene fragments (710 base pairs) of apple polyphenol oxidase (APPO) gene were obtained by RT-PCR amplification, using the total RNAs isolated from ripen apple fruit as the template. These two fragments were ligated by a lOOObp spacer, YYT(crtW+crtY fusion ), gene fragment, which is relative to carotenoid synthesization in subcocci. The full-length 2444bp-target gene was then inserted into plant binary vector pYPX145 to generate the recombinant plasmid pYF7704, which carried the expression unit of APPO dsRNA. Double SAR sequences which is beneficial to the stable heredity of the transgene were located at both ends of the APPO dsRNA expression unit.Through researching on the interior and exterior factors regulating regeneration frequency of apple, we developed a high and stable regeneration system in "red Fuji" apple leaf in vitro. Leaves which located in petiole and nervation after successive subculture for 28 to 35 days had high regeneration frequency. Dark culture was needed in prophase culture, the suitable time of dark culture was about 20 days for red Fuji. Unchanging total nitrogen, instead of the NHjNOa with (NHt) 2864 can enhanced the regeneration frequency of apple leaf in vitro. Different cytokinins exerted their effects on adventitious shoot regeneration Of red Fuji, the hormone composition was TDZ 2mg/L and NAA0.6mg/L.The regeneration frequency is up to 95%, it accords with the transform's request.To develop procedures for the efficient production of apple transgenic plants, we analyzed the principle factors that affect genetic transformation efficiency . In the transformation system of red Fuji, the available concentration of Cb was 100mg/L. The efficient time for explants dipping into Agrobacterium suspension was 5min. Three days co-cultivation and free of CoCl2 -6H2O in co-cultivation for three days medium improved the GUS expression and the explants regeneration . It has beneficial effects on genetictransformation efficiency to decrease kanamycin content in selection medium in steps.pYF7704 was transformed into apple (Malus domesticd) var. Red Fuji via Agrobacterium tumefaciens mediated leaf disc transformation. With the selection of Karamycin and GUS detection assays, transgenic plants of APPO dsRNA were produced. The results of FQ-RT-PCR indicated that APPO mRNA level was suppressed to 91.69% in transgenic plants compared to wild plants. The data suggested that dsRNA technology on the apple polyphenol oxidase is feasible to be utilized in transgenic plants.