Breeding Of Potato Varietris With High Amylopectin And Low Cold-Induced Sweetening Via Transgenic RNAi

With its good features, potato(Solanum tuberosum L.) strach is an important raw material for food and industrial manufacture. Amylopectin takes-80%in total starch in potato tuber. In certain applications, high amylopectin with a lower gelatinization temperature and a slower retrogradation is required. However,-20%of potato starch is amylose, and the separation of amylose from potato starch is energy-consuming.For storage, potato is usually kept about4to10℃to prevent sprout and rot disease, and to prolong the storage. However, the low temperature induces sweetening phenomenon in potato since the starch is degraded into sugars that decrease starch percentage and seriously affect the subsequent processes. High content of sugars will induce the Maillard Reaction, in which the colors of fried potato chips change to black or brown, the chips taste bitter, and acrylamide, a carcinogenic substances, is formed. These changes seriously damage potato quality. Therefore, it is necessary to breed potato varieties with higher amylopectin content. RNA interference is used to silence a gene with endogenous or exogenous double-stranded RNA that is sequence-specific. Small molecules of double-stranded RNA can inhibit the complementary gene expression, causeing post-transcription gene silencing (mainly refers to the siRNA inhibition of the target gene expression in mRNA levels). By regulating the gene expression level, the metabolic activities can be controlled. This technique is usefull and can be applied in potato trait breeding.Based on RNA interference, this study was to down-regulate the Granule-Bound Starch Synthase Ⅰ (GBSS Ⅰ) gene for amylose synthesis, and the Vacuolar Acid Invertase gene (VINV) which plays a key role in cold-induced sweetening in the potato tuber. Totally, three kinds of pHellsgate12vectors containing612bp fragment of GBSS Ⅰ and/or510bp fragment of VINV were constructed for transgenic RNAi that could silence the corresponding genes. RNAi introduced by the vectors successfully silenced both genes, GBSS Ⅰ or VINV, respectively. Using the agrobacterium-mediated transformation, positive lines of transgenic potatos were obtained and verified by PCR. Certain transgenic potatoes were selected in field. qRT-PCR was used to analyze the expressions of GBSS Ⅰ and VINV in the harvested tubers of the transgenic potatoes lines. The silencing efficiency could reach70-90%in most lines. The amylose contents were also tested in the transgenic lines with the highest amylopectin up to99.2%. After low temperature storage, the sucrose content increased slightly, but glucose and fructose were dropped significantly in most tubers of the lines. In the transgenic lines, the colors of fried potato chips were much better than the control. The chip colors of the VINV-silenced lines were better than those of other transgenic lines. Two lines with both silenced genes were excellent in terms of balanced traits of amylose content and fried chip color after low temperature storage.