Construction Of TALEN Plasmid To Modificate Rice OsCENH3 Gene

Haploid angiosperms are sporophytes with gametic chromosome numbers. The first haploid to be reported was found in Datura stramonium in 1921 by A. D. Bergner. Since then, haploid has been playing a very important role in genetic research and crop breeding. In rice,haploid rice comes from natural variation and tissue culture, which has a low efficiency and great workload,is hard to meet the breeder’s requirements. How to produce haploid plant largely and steady is the major concern of breeders. Simon Chan et al discovered a way to produce haploid plant through CENH3 (centromere-specific histone 3) mediated haploid inducer in Arabidopsis in 2010. This promising methods has a high rate of induction and is very convenience and need not tissue culture. The rice OsCENH3 encoded by LOC_Os05g41080,which is single copied in rice genome. The protein CENH3 is evolutionarily conserved and has two major domain:the tail-domain and the histone-fold domain, HFD.In this research,we knock down the native OsCENH3 gene in rice by TALEN and insert a manipulated protein -OsCENH3-tailswap as complementary, aiming to create haploid inducer in rice. The main results are as follows:1. The protein CENH3 is very conserved in different species, mainly in the histone fold domain, but the tail domain is varied. The tail domain of histone 3 and CENH3 is also different even in the same species.2. According to the online chip data, the gene LOC_Os05g41080 encoded the centromere-specific histone 3 in rice is mainly expressed in the SAM and inflorescence.3. In cooperation with a company,we designed a target site of TALEN in the first exon based on the cds sequence of LOC_Os05g41080. We successfully constructed the TALEN plasmid by "Golden-gate" and Luciferase SSA test showed the TALEN pair has a strong activity of gene editing.4. We successfully constructed the complementary fragments ACTIN-GFP-tail(H3)-HFD(CENH3)-NOS using in-fusion technology,and ultimately transferred to the final expression vector PCAMBIA1300 contained TALEN.5. The final vector was introduced into rice embryonic cells using Agrobacterium tumefaciens, and individual transformant cells were selected, propagated and regenerated. We get 249 positive plant, among which, Nipponbare 121, Kittake 128.6. Of 8 randomly selected Nipponbare plants,50%(4) plants showed high expression of GFP by Q-PCR.7. The TALEN target regions of all the positive plants were amplified using the polymerase chain reaction (PCR) and sequenced to detect potential sequence alterations. Unfortunately,.we did not find any mutation in these plant.