[Suppression of apple polyphenol oxidase by double-stranded RNA (RNAi)].
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 with a 1000 bp spacer, YYT (crtW+crtY fusion) gene, which is relative to carotenoid synthesization in subcocci. The full-length 2446 bp-target gene was then inserted into plant binary vector pYPX145 to generate the recombinant plasmid pYF7704, which carried the expression unit, of APPO dsRNA. pYF7704 was transformed to apple (Malus x domestica) var. Red Fuji via agrobacterium tumefaciens mediated leaf disc transformation. With the selection of Karamycin and GUS detection assays, transgenic shoots of A PPO dsRNA were obtained. The results of FQ-RT-PCR indicated that APPO mRNA level was suppressed to 91.69% in transgenic shoots compared to wide shoots. The data suggested that dsRNAi technology on apple polyphenol oxidase is feasible to be utilized in transgenic shoots.
Cao
Yan Hong
YH
Nanjing Agriculture University, Nanjing 210095, China.
Zhang
Zhen
Z
Yao
Quan Hong
QH
Peng
Ri He
RH
Xiong
Ai Sheng
AS
Li
Xian
X
D016960:Agrobacterium tumefaciens / Q000235:genetics; D004156:Catechol Oxidase / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics; D027845:Malus / Q000201:enzymology* / Q000235:genetics*; D008957:Models, Genetic; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D034622:RNA Interference / Q000502:physiology; D012330:RNA, Double-Stranded / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014170:Transformation, Genetic / Q000235:genetics
D018744:DNA, Plant; D012330:RNA, Double-Stranded; D004156:Catechol Oxidase