Construction Of AmiRNA Binary Vector For Candidate Sterile Gene In PTGMS Line HD9802S And Genetic Transformation

The Photoperiod and Thermo-sensitive Genic Male Sterile Lines (PTGMS) rice is an important germplasm resource. The PTGMS gene distributes extensively, expresses differently in variety of PTGMS line. The fertility of Indica TGMS line HD9802S was controlled by one pair of recessive genes and named HDtms temporarily. Previous studies identified HDtms on chromosome2as the locus between the SSR molecular marker RM5897and RMAN8, which contained46.5kb DNA fragments.In this study we concentrate on the following four aspects.1. The46.5kb DNA fragment (No.Scaffold002359) was found in the database of BGI Rise Genome Database, then three unknown genes with7ORF were forecasted in fragment No.Scaffold002359by using Fgenesh software, with which we designed21amiRNA targets.2. Based on the structure of miR528, we constructed an amiRNA vector. In order to accomplish this process, we firstly changed the structure of donor vector (pDONOR-HDtms) by replacing a21bp length sequence on miR528’s stem with the fragment of our target sequence. Then we combined the donor vector (pDONOR-HDtms) with host vector (p1301-gfp) by MAGIC (Mating-assisted genetically integrated cloning).3. We transfered the amiRNA vector into agrobacterium EHA105, and infested cultivated-plants using genetic transformation.4. Positive plants were chosen by PCR, then transplanted to field. The copy numbers of the positive plants were tested by southern blot. Lastly, we observed pollen fertility and analyzed seed setting rates of these transgenic positive plants.We constructed seven amiRNA vectors with comparable genetic modified plants, and the vectors were p528-3, p528-6, p528-8, p528-10, p528-15, p528-18, p528-19, respectively. PCR and Southern blot analysis indicate that the target genes have been successfully transferred into indicia9311. The result of field test shows that p528-8-positive plant is male sterility with low seed setting rate.