Targeting Modification Of BADH2 And Bel Gene Using The Agrobacterium Tumefaciens- Mediates CRISPR /Cas9 System In Rice

Rice is one of the most important food crops in the world, and provided food with more than half of the population in the earth, what's more, it also is the main food crop in southern of China. But with the international agricultural markets for agricultural products and the improvement of people's quality of life, more and more people tend to eat rice with aromatic,such as Thai fragrant rice, Jasmine rice. In addition, hybrid rice is the main planting rice in southern of China, but the production process of hybrid rice is due to the intercropping of male sterile line and restorer line, which may affect the purity and mechanization level of hybrid rice. Therefore, the selection and cultivation of herbicide sensitive restorer lines is benefit to reduce the labor costs of hybrid rice seed production greatly and improve the level of hybrid rice production mechanization.In this paper, the BADH2 gene and the Bel gene of rice were screened with bioinformatics and edited by the new genome editing technique CRISPR / Cas9, which was developed rapidly in recent years. We obtained a series of transgenic plants by agrobacterium-mediated genetic transformation. Furthermore, the traditional PCR technique,digestion with restriction enzyme and sequencing were used to detect and found insert and other mutations. The targeted modification system for CRISPR / Cas9 system and the process of rice genetic transformation were preliminarily mastered via this experiment.The main results as follows:(1 ) Construction of target-sgRNA-Cas9 recombinant vector: Using means of bioinformatics and genetic engineering, three targets were selected from BADH2 gene and three targets--sgRNA-Cas9 recombinant vector were fabricated, marked as pCAMBIA1301 -fgr-sgRNA 1 -Cas9?pCAMBIA1301 -fgr-sgRNA2-Cas9 and pCAMBIA 1301 -fgr-sgRNA3-Cas9; similarly, one target was selected in the Bel gene and the pCAMBIA1301-ben-sgRNA1-Cas9 recombinant expression vector was also constructed.(2) Construction of agrobacterium recombinant engineering: Agrobacterium tumefaciens expression system containing pCAMBIA 1301-fgr-sgRNA1-Cas9?pCAMBIA 1301-fgr-sgRNA2-Cas9?pCAMBIA1301-fgr-sgRNA3-Cas9 and pCAMBIA1301-ben-sgRNA1-Cas9 were obtained by transformation with electroporation transformation.(3) Obtained transgenic plants in rice: Transformation and induction of rice callus:agrobacterium tumefaciens-mediated transformation was used to transform recombinant agrobacterium tumefaciens into TP309 callus. After screening, induction and differentiation,rice seedling plants were obtained. Then, the DNA of rice was detected by PCR and electrophoresis, and we found there were 4 transgenic rice plants of pCAMBIA1301-ben-sgRNA1-Cas9, 21 transgenic rice plants of pCAMBIA1301-fgr-sgRNA1-Cas9 transgenic rice plants and 34 transgenic rice transformants of pCAMBIA1301-fgr-sgRNA2-Cas9 and 29 transgenic rice plants of pCAMBIA1301-fgr-sgRNA3-Cas9.(4) Target site mutation detection: The target site sequence in the genome was identified by PCR, restriction enzyme digestion and sequencing. It was found that 9 of the 34 loci of the target site fgr-sgRNA2 had a site-directed mutation, and the target mutation rate was 26%when the target site fgr-sgRNA3 have about 31% mutation efficiency. Nevertheless, target site ben-sgRNA1 only found 2 mutations while fgr-sgRNA1 did not detect a mutation.What's more, the mutations of target site fgr-sgRNA2 mainly occurs base replacement while target site fgr-sgRNA3 is based on single base insert, similarly, target site ben-sgRNA1 occurs insert and replace. From the overall mutation type analysis, the insert mutation was about half of the proportion of mutations in this paper.(5) Phenotypic analysis: The KOH method and the sensitivity test were used and found that some plants were distributed fragrance with the leaves. What's more, in the transgenic plants of Ti generation ben-sgRNA1, a susceptible plant was found.