Genome Engineering And Interference Study Of Panicle-specific Expressed Genes In Rice

Rice(Oryza sativa) is a staple food for over half of the world’s population. Panicle type plays a leading role in optimal design of plant type and cultivation for high-yield breeding. Mutant is one of the most strait-forward and efficient way to study gene function. Aiming to study panicle-specific genes’ function, we selected six panicle-specific genes based on gene chips data. Using CRISPR/Cas9 technology; over-expressor gene hunting technology; RNA Interference(RNAi) technology and chimeric repressor silencing technology(CREST), we generated these genes’ mutants. The main results were described as follows:1. Analysis of gene chips data showed that it was expressed specifically in panicles that auxin efflux transporter gene Os PIN1 c and TCP family genes PCF5, PCF6, Os TCP7, PCF8 and Os TCP9. Analysis of gene expression profiles proved that these genes were expressed specifically in young panicles by q PCR. It proved the reliability of gene chips data. The expression patterns of gene may relate to its function.Thus we concluded that those genes would probably involve in the control of rice panicle type.2. Using CRISPR/Cas9 technology, we constructed the single-gene targeted CRISPR/Cas9 transgenic plants of the six genes and multi-targets CRISPR/Cas9 transgenic plants of PCF5 and PCF6. Molecular analysis of the transgenic plants showed that the genes were edited successfully. We obtains mutants of the six genes and double homozygous mutants pcf5 pcf6. Compared with wild-type, all mutants had no significant phenotypic variation in panicles. These proved that the monogenic mutations of the six genes and the multi-gene mutations of PCF5 and PCF6 do not lead to significant phenotypic variation in rice. Besides, about half of multi-targets CRISPR/Cas9 transgenic plants of Os PIN1 c gene appeared to the mutant phenotype as lax panicle. However, the mutant phenotype of the T1 line was not co-segregation with the genotype of Os PIN1 c locus. It proved that the mutations of a novel gene may affect the growth and development of panicle in rice.3. To further study the function of these five TCP genes, we constructed their other types of mutants. We constructed these genes’ over-expression vectors, SRDX vectors and RNAi vectors. And then we obtained transgenic plants by Agrobacterium-mediated transformation method. The positive test results showed that the positive rate was more than 80%, which indicated transgenic efficiency was high. q PCR analysis showed that changes in the relative quantitative expression of these genes were in line with expectations. The results of phenotype observation indicated that compared with wild-type, all TCP transgenic plants had no significant phenotypic variation.