Study On The Gene Of PTGMS2-1 Directed Mutation By CRISPR/Cas9 Technology In Rice

Rice is one of the most important food crops in the world, and hybrid rice breeding is an important way to increase rice production. Currently the most effective method is to selectively breed photoperiod-temperature-sensitive sterile lines to combine hybrid rice.However, there has been no particularly effective selection path so far due to various restrictions of the selection and breeding of photoperiod-temperature-sensitive sterile lines. CRISPR/Cas9 mediated gene editing techniques is another gene editing technique besides zinc finger nuclease(ZFNs), transcription activator-like effector nucleases(TALENs) and meganucleases technology. It offers a very effective way of gene mutation technology for improvement of target gene due to its high targeting efficiency, short cycle,simple vector structure, etc. With the promising development of functional genome of rice,a group of photoperiod-temperature-sensitive sterile genes have been cloned, laying very good foundation for directional mutation. In this study, rice recessive temperature-sensitive sterility genes( PTGMS2-1) were selected to acquire temperature-sensitive rice sterile lines by directional mutation of CRISPR/Cas9 technology. The researches below were carried out with results as follows.1 、 Constructing CRISPR/Cas9 directional mutation vector. After analysis of gene sequence of PTGMS2-1, the target sequences of 20 bases with A beginning to NGG ending and G beginning to NGG ending were selected on exon. And, while the sequences with A beginning were connected to the binary vector containing U3 promoter, the sequences with G beginning were connected to the binary vector containing U6 promoter.The transformation method of e. coli was utilized to screen the target vector, whose sequencing and enzyme digestion verified the success of the connection of target sequences and the integrity of the vector.2、To acquire the transgenic plants. The binary vectors were induced into the callus of indica yue tai B through agrobacterium transformation method. 14 transgenic plants were acquired after plant tissue culture, with 8 from U3 promoter mediation and 6 from U6 promoter mediation.3、Testing transgenic plants. PCR detection indicated that the 14 transgenic plants all were positive ones. The HMR was used to preliminarily analyze the mutation type of T0 plan.The results showed that mutation was found in 5 plants among the 8 positive plants from U3 promoter mediation, indicating heterozygote, and that mutation was found in 3 plants among the 6 positive plants from U6 promoter mediation, also indicating heterozygote.