Production And Phenotypic Study Of Knockdown And Knockout Plants Of Rice DNA Methyltransferase Genes CMT2/3

DNA methylation is a conserved and important epigenetic modification.DNA methylation can silence transposable elements(TEs)and repetitive sequences and repress transcription.DNA methylation mainly occurs in CG,CHG,and CHH sequences(H represents A,T and C)in plants.CMT2 and CMT3 DNA methyltransferases are plant specific;they can maintain non-CG methylations.We mainly studied the function of rice OsCMT2,OsCMT3 a,and OsCMT3 b genes in DNA methylation.The major results are as following:1.We obtained 30 OsCMT2 RNAi lines and analyzed the expression level of OsCMT2 in these lines.No phenotype was observed in the highly repressed lines.We also identified T-DNA insertion mutants of Oscmt2,and backcrossed these mutants with WT twice to eliminate transgenic procedure-induced mutations.The mutants show decreased pollen fertility.We constructed Oscmt2 mutant lines in the HY background by CRIPSR-Cas9.The CRISPR-Cas9 mutants showed an etiolating phenomenon and early earing.2.We obtained 24 OsCMT3 a RNAi lines and analyzed the expression level of OsCMT3 a in these lines.We also studied the phenotype(pollen fertility and agronomic traits)of the lines with high level repression of the gene.Six transgenic lines with strong phenotypes were selected for further BS-seq analysis.We constructed Oscmt3 a mutants in the DJ background by the CRIPSR-Cas9.The mutants show severe developmental defects and infertility.3.We obtained Tos17 insertion mutants of Oscmt3 b,and backcrossed them with WT to eliminate transgenic procedure-induced mutations.The mutants showed delayed heading and reduced seed setting rates.We also constructed Oscmt3 b mutants in the DJ background by CRISPR.The mutants showed shorter panicle and plant height.