Establishment And Application Of High Efficiency Rice Gene Silencing System Based On MIGS

As the one of most important foodstuff in the world, rice is popular in almost one second of global people. However, with increasing of the population and decreasing of the arable land, the higher yield and quality of rice are very urgent. With the completion of rice genome sequencing, functional genomics has become the focus of the research work in rice. More and more technical methods of ana lysis and identification of gene function is appeared. The RN A interference(RNAi) as an important method of reverse genetics research has been widely used in gene function study.There exists a special kind of micro-RNAs(miRN As)called trans-acting siRNA(tasiRNA)in plants. tasiRNA can induce a series of small interfering RNAs(siRNA)which resulting in gene silence. Many miRNAs namely miR173, miR390 and miR828 are involved in the biogenesis of tasiRNAs. The miR173 mediated ta-siRNA formation has been applied recently for the development of miRN A mediated gene silencing(MIGS) vector for silencing of target genes.MIGS was firstly reported in 2012 and it can effectively mediated target gene silencing in in Arabidopsis thaliana. To date, MIGS hasn’t been effectively updated and applied. Moreover, there are no reports about whether MIGS can be used in the monocotyledon. In this research, based on the mechanisms of tasiRN A and MIGS, the expression patterns of monocotyledon, we constructed the MIGS backbone vectors including p ZHY930, pZHY931 and pZHY932 based on different expression patterns of miR173 and miR173_ts. Firstly, we select the rice OsPDS as a reporter gene to obtain the transgenic albino rice through genetic transformation. The results exhibited that all three backbone vectors can induce the target gene silencing, producing the albino rice. In the albino rice, the RNA level of OsPDS was significant decreased. To our surprise, the silencing efficiency of p ZHY932 was as high as 90%. Then we selected Os ROC5 and OsLZAY1 as anthor two endogenous genes of MIGS silencing, and we abtianed the mutated rice successfully. What’s more, we using pZHY932 as the backbone vector constructed double gene sile ncing vectors which can silent OsPDS and Os ROC5 or OsPDS and OsLZAY1 at the same time. And we got the double mutated rice with albioned and rolled leaves or albioned and tiller angle increasing leaves. At last we established the MIGS efficient gene silencing system in rice.Based on the MIGS technology system, we constructed MIGS-OsGBSS vector and transformed in rice. Using our MIGS technology, we can high amylopectin starch by regulate the expression of starch synthesis in rice.In conclusion, in this stud y we not only constructed the MIGS backbone vector, MIGS- OsPDS, MIGS-Os ROC5, MIGS-OsLAZY1, MIGS double silence vector and MIGS-OsGBSS vectors based on MIGS system, but also achieved the effective silencing of target gene in rice. We provide an effective theoretical basis for the further application of MIGS in monocotyledonous plants.