The Silencing Of Polyphenol Oxidase Genes In Potato Triggered By RNA Interference

Polyphenol oxidase (PPO) is the major cause of enzymic browning in potato. In vivo, the PPO localized in plastids, while the phenolic substrates of PPO are localized in the vacuole. The browning reaction only occurs as a result of tuber damage leading to a loss of this subcellular compartmentation. PPO catalyzes the conversion of monophenols polyphenols to o-dihydroxyphenols or to quinones. The quinone products can then polymerize and react with amino acid groups of cellular proteins, resulting in brown or black pigment deposits. Such damage causes considerable economic and nutritional losses in the process of potato.Recent work has focused on designing the constructs which require two copies of the target sequence in an inverted-repeat orientation encoding self-complementary `hairpin' RNA with a intron between the two copies (ihpRNA), in order to produce duplex RNA to efficiently silence the genes in vivo (called RNAi technology). RNAi technology allows silencing all members of a multigene family or homoeologous gene copies in polyploids by targeting sequences that are unique or shared by several genes.In this study, we cloned one member of PPO genes-POT33 and constructed a constitive RNAi vector promoted by 35S promoter carrying two copies of part sequences of POT33 in an inverted-repeat orientation (called pMBW330-ihpPOT33), and a tissue-specifical RNAi vector promated by GBSS carrying two copies of part sequences of POT33 in an inverted-repeat orientation (called pGB121s-ihpPOT33). Agrobacterium LBA4404 harboring the binary vector pMBW330-ihpPOT33 or pGB121s-ihpPOT33 was employed to transform Potato 'Atlantic', and 75 potato plantlets were regenerated after transformation. The transformed potato plants were confirmed by PCR and Western hybridization successfully. The results suggested that one member of the target protein was silenced in two transformed potato plants, and the silencing efficiency of them was 86.02 % and 66.01 % analyzed by software Bandscan, respectively.