Functional Study Of Rice Chromosome Structure Maintenance Proteins SMC 1 And SMC3 In DSB Repair

SMC proteins maintain the morphological characteristics of chromosomes.There are six SMC homologous proteins in eukaryotes:SMC 1-6.SMC proteins mainly function in the form of complexes.Six kinds of SMCs in eukaryotic organisms form heterodimers(SMC1 and SMC3,SMC2 and SMC4,SMC5 and SMC6),and then combine with other non-SMC protein components to form three kinds of SMC protein complexes(condensin,cohesin and SMC5-SMC6 complex).DNA double strand breaks(DSB)are highly cytotoxic DNA damage and will severely threaten the physical and genetic integrity of genome if not properly repaired.DSB can occur during DNA replication or caused by DNA damage factors.The correct repair of DSB is the basis of cell survival and genomic stability.There are two main mechanisms of DSB repair in eukaryotic cells:non-homologous end-joining(NHEJ)and homologous recombination(HR).HR can be divided into two types according to whether chain invasion is catalyzed by RAD51 recombinase during repair:synthesis-dependent strand annealing(SDSA)pathway that requires RAD51 recombinase and single-strand annealing(SSA)pathway that does not depend on RAD51 recombinase.Studies in yeast and Arabidopsis have shown that SMC1 and SMC3 proteins are involved in the repair of DSB,but the specific mechanism is still unclear.However,their roles in DSB repair in rice have not been studied.In this study,we have made a preliminary study on the functions of SMC1 and SMC3 genes in rice.The main results are as follows:1.We successfully constructed vectors for editing target genes using CRISPR/Cas9 CRISPR/Cas9 system and obtained transgenic plants.2.Real-time PCR analysis of SMC1 and SMC3 expression in different tissues of rice showed that SMC1 and SMC3-1 were highly expressed in young panicles of rice,while SMC3-2 and SMC3-3 were highly expressed in leaves.3.Frameshift mutations in SMC1 and SMC3-1 genes cause death in rice embryos.4.Mitomycin sensibility tests indicated that SMC1 and SMC3-3 genes might be involved in the biological event of sister chromatid adhesion in rice5.Sensitivity tests showed that smc1 and smc3-2 mutants were more sensitive to Bleomycin.6.Real-time PCR results indicated that SMC1 and SMC3 affect the repair of DSB through HR or NHEJ pathway.7.The results of Immunization showed that the recruitment of RAD51A2 in smc3-3 was significantly lower than that in the wide type,indicates that SMC3-3 participates in DSB repair through the SDSA.8.The results of recombination showed that the mutations in SMC1,SMC3-2 and SMC3-3 genes could reduce the repair rate of SSA in rice somatic cells,but only in smc3-3 SDSA repair pathway was found to be affected.That is to say,SMC1 and SMC3-2 participate in the process of SSA,while SMC3-3 also participates in the process of SSA and SDSA.