New Potato Germplasm Resistant To Browning Was Developed By RNAi And Gene-Deletor Techniques

Potato（Solanum tuberosum L.）is an annual herb of Solanaceae.It is one of the four major food crops in the world and one of the important cash crop in China.However,potatoes are prone to enzymatic browning during processing,which significantly reduces their appearance quality and nutritional value,severely restricting the development and utilization of potato products.Solving the browning problem is of great significance for the development of the potato industry.Studies have shown that polyphenol oxidase（PPO）in potatoes is a key enzyme that causes browning in potatoes.This study aims to obtain a new potato germplasm that is genetically safe and has strong browning resistance.Using the conserved domain Tyrosinase in the St POT32 gene as the interference target,an RNAi expression vector containing Gene Delta elements was constructed.Through Agrobacterium mediated genetic transformation of potato variety Desire,the effect of RNAi interference with PPO genes on the browning resistance of potato tubers was studied,and the deletion rate of foreign genes in tubers was analyzed.This provides a new technical approach for creating new germplasm that meets the safety of transgenic technology.The following results were achieved:1.Construction of St PPOi Vector and Genetic Transformation of PotatoesThe conserved domain Tyrosinase fragment of St POT32 gene was cloned from the tetraploid cultivar Desiree.With this conserved domain as the interference target,the tuber specific promoter patatin drives the target gene,and p9286 containing Gene-Deletor element as the starting vector,which is a combination of FLP/FRT system of yeast and Cre/Ox P system from bacteriophage,a fusion recognition site LF（Lox P FRT）of lox P and FRT was created,We obtained an efficient gene clearance system and selected a specific cold induced tuber fusion promoter to drive the Gene-Deletor system.We successfully constructed an RNAi expression vector with foreign gene deletion function,named p9286-St PPOi.Through Agrobacterium mediated genetic transformation,16 positive potato transgenic plants with PPO gene silencing were obtained.2.Effect of RNAi interference with PPO gene on potato browning phenotypeUnder room temperature conditions（25±1℃）,freshly cut potato tubers were exposed to air and observed.As the exposure time increased,the cut surface of wild type and p9286 transgenic potato tubers gradually showed brown at 3 hour,while transgenic potato tubers began to appear brown at 12 hours.The change first starts in the ring bundle area of the vascular system,and then gradually spreads and extends to the central medulla.At 24 hours of tuber aging,the browning grading was statistically and calculated.The browning index of wild-type was 34.44%,the browning index of transgenic plants was 35.55%,and the browning index of transgenic plants was16.67%.The browning index of wild-type and transgenic plants was higher than that of transgenic plants,and the difference reached a very significant level（P<0.01）.The results showed that the brown color of transgenic potato tubers appeared later than that of wild-type and empty vector potato tubers,and the browning development speed was slow,with a relatively light degree of browning.3.The effect of RNAi interference with PPO genes on PPO enzyme activityThe PPO enzyme activity of potato tubers was measured using the polyphenol oxidase enzyme activity assay kit,and the enzyme activity of wild type potato tubers was 133.8(U·g^-1);The potato tuberase activity of the empty vector was 131.2(U·g^-1);The enzyme activity of transgenic potato tubers p9286-St PPOi was 56.6(U·g^-1),The PPO enzyme activity of transgenic potato tubers decreased by 56.8% compared to that of transgenic plants,and decreased by 57.7% compared to that of wild type plants,with significant differences（P<0.01）.The decrease in PPO enzyme activity delayed the occurrence of browning and reduced the browning index.4.Effect of RNAi interference with PPO gene on its relative expression levelqRT-PCR analysis of potato tubers showed that the expression levels of PPO genes in the transgenic lines decreased to varying degrees compared to the empty vector and wild-type lines,indicating that p9286 St PPOi had been integrated into the potato genome for normal transcription and interference,thereby inhibiting the enzyme activity of PPO.5.Statistics of foreign gene deletion efficiency in transgenic potato tubersAfter cold induction of transgenic potato tubers at 2℃ for 3 days,a foreign gene deletion system was activated.GUS gene histochemical staining was performed on potato tubers,and it was found that some transgenic potato tubers were not dyed blue.Primers were designed using GUS gene,and PCR validation showed that no foreign genes were detected in 8 out of 16 transgenic potato tubers,with a deletion rate of50%;Out of the 16 empty potato tubers,7 of them did not detect foreign genes,with a deletion rate of 43.75%.However,the potato tubers treated at room temperature（25±1℃）all detected the presence of foreign genes.Successfully obtained a new potato germplasm that does not contain foreign genes and has strong browning resistance.