Preliminary Inquiry Study Of Engineering Nuclear Male Sterility System In Oryza Sativa

Abstract:Rice is one of the major staple foods in China, and hybrid rice is regarded as " the fifth great invention" of China. Heterosis utilization is one of the core technologies for food security system in China. The "three-line system" and "two-line system" are two convetional ways for heterosis utilaization. However, due to a lack of useful parental resources and existing security concerns in seed production, these two methods both have shortcomings in utilizing the full potential of heterosis. The nuclear male sterility provides an ideal method for heterosis utilization, advantages include a wide variety of sources, unlimited potential in line maintainance and restoration, thorough recessive gene abortion with little environmental impacts and etc. But the main obstacle in commercial applications of nuclear male sterility lines in the propagation of male sterile lines, which limits the wide-spread adoption of this method for commercial use.Here, we address conventional nuclear sterility propagation problems through genetic engineering methods. By constructing a functional "third-generation hybrid rice" application system, we developed provisional father lines that carry a TDR fertility restoration gene and EGFP expression driven by an endosperm-specific promoter PGt1and obtained29positive strains of genetically modified rice. We also constructed a chloroplast genetic transformation vector which contains three cistrons consisting of11gene fragments from5species. The prokaryotic expression was further verified with green fluorescence protein in E.coli. These tools provide a foundational component to chloroplast genetic ways of nuclear sterility system. Moreover, we established a new effective and efficient method for plasmid vector construction through recombinant-fusion PCR method. With this method several fragments can be fused to create a complete construct in a single step, providing a convenient tool for the follow-up study and reducing the difficulty of multi-gene fragment vector construction.