The Functional Studies Of TOP6,SMC1/3 And MiRNA In Rice Somatic DSB Repair

Rice is an important food crop,which provides the staple food for more than 3.5 billion people and is of great significance to the development and survive of human.The problems that decrease of cultivated land and the harm of various environmental presses have seriously threatened the security of rice production.Under increasingly severe environmental conditions,it is very important to ensure high quality and high yield of rice.The study of DNA damage repair mechanism is helpful to understand the molecular mechanism of maintaining genomic stability in rice,and can provide gene resources and theoretical basis for genetically improving crop stress resistance and crop yield.Organisms is subjected to various DNA damage caused by endogenous and exogenous factors all the time,among which double strand break(DSB)is a highly harmful form of damage.If DSB can not be repaired effectively,it can cause death of the organism.There are two major DSB repair mechanisms in eukaryotic cells:canonical nonhomologous end joining(c-NHEJ)and homologous recombination(HR).Based on whether RAD51 recombinase is required,HR repair can be divided into two types:synthesis-depende strand annealing(SDSA)and single strand annealing(SSA)pathway.DNA topoisomerase exists in eukaryotic cells and prokaryotic cells and plays a role of changing DNA topological state in many biological processes.DNA topoisomerase is classified into two types:type ? and type ?,according to their different ways of topological modes.DNA topoisomerase ?(TOP6),a heterotetrameric complex composed of two TOP6A and two TOP6B subunits,belongs to a novel family of type ? DNA topoisomerases present.TOP6 is involved in altering DNA topology during replication,transcription and chromosome segregation by catalyzing DNA strand transfer through transient double-strand breaks.Chromosomes are not only composed of DNA,but also contain some proteins that play a role in gene expression and genomic stability.Among them,SMC(Structural maintenance of chromosomes)is the primary substance that enables chromosomes to have specific morphology.There are six SMC homologues in nuclear organisms:SMC 1-6.The SMC1 and SMC3 cohesin complex are mainly involved in sister chromatid cohesion,DNA repair and checkpoint activation in the cell cycle.MicroRNA(miRNA)is a 20-24 nucleotide(nt)long non-coding RNA,which is processed from primary miRNA(pri-miRNA)transcript containing stem loop of secondary structure.It can regulate gene expression through the silencing mechanism of degrading the target genes of complementary sequence.They have been reported to be involved in the DNA damage response(DDR)mechanism in other species,but whether they still have the similar role in rice and what is their molecular mechanism remain unclear.In this study,TOP6,cohesin proteins(SMC1/SMC3)and miRNA were used as entry points to explore their functions and mechanisms during the repair of somatic DNA damage in rice through comprehensive use of physiology,transcriptome,cytology and other molecular biological methods.Part ?:Functional study of rice chromosome structure maintenance proteins SMC1 and SMC3 in DSB repairThe SMC1/SMC3 heterodimer acts in sister chromatid cohesion,and recent data indicate a function in DNA double-strand break repair.Since this role of SMC proteins has remained largely elusive,we explored interactions between SMC 1/3 and the factors in HR or NHEJ pathways for DSB repair in rice.In this study,rice smc1 and smc3 mutant plants were obtained through CRISPR-Cas9 technology.Through bleomycin,mitomycin,radiation treatment and real time quantitative PCR(RT-qPCR)analysis,we found that SMC1 and SMC3-1 genes may be involved in DSB repair and mitosis in rice.Root tip immunostaining and recombinant rate analysis confirmed their function in DSB repair and identified the repair pathway involved.The main results are as follows:1.Frameshift mutants of smcl and smc3-1 can not be obtained.Non-frameshift mutants of smcl and smc3-1 are survive in normal condition.All frameshift mutant lines of smc3-2 grew normally.2.SMC1,SMC3-1 and SMC3-2 genes were expressed in all tissues of rice,among which SMC1 and SMC3-1 genes had the highest expression in young panicle,while SMC3-2 genes had the highest expression in leaves.3.Subcellular localization in rice protoplasts showed that SMC1,SMC3-1 and SMC3-2 were located in cytomembrane and nucleus.4.Both smc1 and smc3-1 mutants showed resistance to mitomycin,while smc3-2 mutant did not show significant resistance to mitomycin.We hypothesized that SMC1 and SMC3-1 genes may play important roles in the sister chromatid cohesion events in rice.5.After bleomycin or ?-ray treatment,the root length of smc1 and smc3-1 mutants were significantly shorter than that of the wild type,while the root length of smc3-2 mutant did not change significantly.Based on this analysis,we considered that SMC1 and SMC3-1 genes were involved in the mechanism of DDR(DNA damage response)in rice.6.RT-qPCR analysis of DSB repair genes after bleomycin and irradiation(IR)treatment showed that compared with the wild type,the expression levels of major SDSA pathway genes were significantly lower than that before treatment in smc1 and smc3-1 mutants.The expression levels of most SSA pathway related genes in smc1 and smc3-1 mutants were the same as those of the wild type.However,compared with the wild type,the expression levels of major C-NHEJ pathway genes were significantly higher than that before treatment in smc1 and smc3-1 mutants.These results suggest that SMC1 and SMC3-1 genes can regulate the expression of genes related to SDSA of HR repair pathway and c-NHEJ pathway.7.After 40 Gy IR treatment,the immune signals of RAD51 and XRCC3 proteins were significantly reduced in smc1 and smc3-1 mutants,while those of RAD51B,RAD51C and LIG4 did not change significantly.These results indicated that SMC1 and SMC3-1 genes affected the recruitment of SDSA related proteins.8.Recombinational rate analysis showed that compared with the wild type,the SSA repairs(PCR bands)could be detected in both smc1 and smc3-1 mutants.On the other hand,the SDSA repairs(PCR bands)were greatly compromised in smc1 and smc3-1 mutants,compared with the wild type.This indicated that smc1 and smc3-1 mutants only reduced the repair efficiency of SDSA,while SSA repair efficiency did not change significantly.Part ?:Functional analysis of rice TOP6A3 and TOP6B in rice somatic DSB repairStudies in Arabidopsis and rice have identified multiple TOP6A homologues and one TOP6B homologue.It has been found that some of TOP6 genes play important roles in the formation of DSB in meiosis,but whether they also play roles in the response to DSB damage in somatic cells remains unclear.In order to understand the function of rice TOP6 genes in the somatic DSB repair,we conducted a series of experiments using their over expression and CRISPR-Cas9 lines in this study.The main results are as follows:1.The mutant plants of top6a3 and top6b grew slowly at seedling stage and died after 1 month.The heading date of TOP6A3 OE and TOP6B OE was obviously advanced.2.TOP6A3 and TOP6B genes were expressed in all tissues tested.3.Subcellular localization assay of TOP6A3 and TOP6B in rice protoplast showed that they were localized in the nucleus.4.Under bleomycin treatment,TOP6A3-OE and TOP6B-OE plants grew more slowly than wild type.After 100 Gy IR,the chlorophyll content of TOP6A3-OE and TOP6B-OE plants were significantly reduced compared with the wild type,which proved that TOP6A3OE and TOP6B-OE plants were very sensitive to genotoxicity in rice.5.RT-qPCR and fluorescence quantitative analysis indicated that bleomycin strongly induced the expression of HR genes,but slightly inhibited the expression of c-NHEJ genes.On the contrary,the expression of HR genes were inhibited by salt stress,while the expression of c-NHEJ genes were up-regulated.Interestingly,transcriptional levels of genes in both pathways were significantly increased under cold stress.These results indicate that the expression patterns of DSB repair genes activated in rice cells are different under different stresses.6.Under bleomycin treatment,the expression levels of HR-related genes in ku70,ku80 and com1 mutants were significantly up-regulated.On the other hand,the transcription levels of c-NHEJ-related genes in the mutants related to HR pathway(rad51c,rad51d,and rad51a1rad51a2)were also significantly enhanced after treatment with bleomycin.These results suggest that there is a reciprocal transcriptional inhibition relationship between HR and c-NHEJ factors in rice somatic cells.7.Under 40 Gy IR treatment,the expression levels of HR pathway genes(RAD51A2,BRCA1,RAD51B and RAD51C)were increased in TOP6A3-OE and TOP6B-OE plants compared with WT,while the transcription levels of c-NHEJ genes(KU70,KU80 and LIG4)were decreased to varying degrees.In addition,after 40 Gy IR treatment in NIP plants,chromatin immunoprecipitation(ChIP)assay show that TOP6A3 interacted with the promoter regions of KU70,KU80,BRCA1 and RAD51A2 genes with TOP6A3 antibody.These results suggested that TOP6 complex could directly positively regulate the transcriptional expression of HR pathway genes(RAD51A2,BRCA1)and negatively regulate the transcriptional expression of c-NHEJ pathway genes(KU70,KU80)during DNA damage repair in rice.Part ?:Deep Sequencing Discovery and Profiling of miRNAs Produced in Response to DNA Damage in RiceIn this study,we use bleomycin to simulate extreme environmental stress to induce DNA damage in rice seedling roots.Differentially expressed miRNAs were identified by highthroughput sequencing of bleomycin-treated and non-treated control groups.The corresponding target genes of differentially expressed miRNAs were predicted.We integrated RT-qPCR,Gene ontology(GO)enrichment and Kyoto encyclopedia of genes and genomes(KEGG)pathway to analyze the potential regulatory mechanism of miRNA's extensive involvement in rice DNA damage repair.To better understand the roles of miRNAs in rice DNA damage repair,we constructed two small RNA(sRNA)libraries of rice roots with or without bleomycin treatment.We identified a total of 513 known miRNAs and 72 novel miRNAs in two libraries using Illumina Hiseq2000 sequencing technology.A total of 140 known miRNAs and 10 novel differentially expressed candidate miRNAs were identified that may have important roles in response to bleomycin-induced DNA damage in rice.A total of 8731 corresponding target genes were predicted as targets of these miRNAs.The GO enrichment analysis of these target genes indicated 41 target genes were involved in DNA repair processes.We confirmed the sequencing results of differentially expressed miRNAs and their potential target genes via RTqPCR.While the exact functions of these differentially expressed miRNAs requires further investigation,the findings of this study provide valuable information for a further functional analysis of the miRNAs produced in response to DNA damage in rice.