Studies On The Biological Function Of Mre11 Gene In Rice(Oryza Sativa L.)

Meiotic recombination 11(Mre11)is a nuclease protein whose structure and function are relatively conserved in various species.In yeast,humans and Arabidopsis,it has reported that Mre11 participates in the process of meiosis and plays an important role in the repair of DNA damage.In rice,it is found that OsMre11 is involved in the process of meiosis.However,the role of this gene in the DNA replication and damage repair during rice mitosis is unclear.In this study,using the mre11 mutant as the research material,by using rice transgenic technology,transparent of root tip,transparent of ovary,pollen grain staining,fluorescent quantitative PCR,flow cytometry analysis,comet experiment,Bi FC,Co-IP and other cell and molecular biological methods,we revealed that Mre11 plays an important role on DNA replication and DNA damage repair of the vigorously dividing root tip meristem cells to maintaining the normal growth and development of plants in rice.Furthermore,the interaction mode of the rice MRN complex was also explored and studied.The main research results obtained were as follows.1.Mre11 gene mutation affected the normal growth and development of rice.The T-DNA insertion mutant of the Mre11 gene was obtained from the RISD DB of the rice mutant library.Sequencing analysis revealed that the T-DNA in homologous was located in the 19 th intron of DNA.Phenotypic observation revealed that the mre11 mutant was short and the seeds were completely aborted.The complementary experiment showed that the phenotype of the mutant could be restored,and the CRISPR/Cas9 gene knockout experiment yielded another mutant mre11-cr that was similar with the T-DNA insertion mutant in phenotype,indicating that the mutation of OsMre11 gene was the important reason of dwarf plants and seeds abortion.The genetic segregation ratio results showed that wild-type: heterozygous: homozygous was1:2.28:0.61,indicating that mre11 is a single-gene recessive genetic mutant.Observation of ovary transparency and pollen grain staining of osmre11 mutant showed that the embryo sacs of homozygous plants were completely shrunk and all pollen grains were aborted,indicating that the abnormal development of male and female gametophytes was the key factor for seed abortion.Using CRISPR/Cas9 gene knockout technology to genetically modify the corresponding genes of Rad50(Radiation sensitive50)and Nbs1(Nijmegen Breakage Syndrome 1),combining with Agrobacterium tumefaciens-mediated rice transgenic technology for genetic transformation,we obtained homozygous mutants rad50-cr and nbs1-cr,but these two mutants had no influence on plant growth and development.2.OsMre11 was involved in the process of mitosis.Through observating the transparent root-tip of mre11 homozygous seedlings from seed-germinating for 7 days,it was found that the length of the meristem of mre11 was shorter than that of wild-type,the cell size and the number of cells was reduced.In addition,through observation of the root tips with pressing and staining methods,we found that a large number of chromosome fragments and bridges appeared in the metaphase and anaphase of mitosis in mre11 and mre11-cr root tips.Among them,the chromosome abnormality ratios in the root tip of mre11 reached 25.86% and 34.78% in mre11-cr,indicating that the OsMre11 gene mutation seriously affected the mitotic process of cells.3.OsMre11 was a widely expressed gene.The expression of OsMre11 in rice different tissues was detected by using q PCR technique and the promoter fusion GUS expression vector.The results showed that Mre11 expressed in different tissues of rice,and the expression level in young tissues was the highest,indicating that OsMre11 might play roles in rice growth and development,and was more important in the dividing tissues.The transient expression analysis showed that OsMre11 was localized in the nucleus,indicating that it was a nuclear localization protein.The expression levels of OsRad50 and OsNbs1 were significantly different from OsMre11,indicating that the three genes have different expression patterns,and implying that they might have different functions and participate in other biological events.4.OsMre11 participated in DNA replication.Different concentrations of replication inhibitor hydroxyurea(HU)and Aphidicolin were used to treat wild-type seedlings germinated for 10 days.The quantitative testing results showed that OsMre11 was very sensitive to HU.At the same time,Ethynyl deoxyuridine(Ed U),a specific dye for the S phase of the cell cycle,was used to stain wild-type and mre11 root tip cells.The results showed that the number of Ed U-labeled cells in the root tip of the mre11 mutant was significantly less than that of the wild-type,and their number of Ed U-labeled cells accounted for the proportion of the total number of meristem cells was also significantly decreased,indicating that the OsMre11 gene mutation severely inhibited DNA replication in the cells of root tip meristem of seedlings,suggesting that OsMre11 was a key factor in the process of mitotic DNA replication.5.OsMre11 was involved in DNA damage repair.Different concentrations of DNA damaging agents mitomycin C(MMC)and methyl methanesulfonate(MMS)were used to treat wild-type seedlings germinated for10 days.The quantitative testing results showed that OsMre11 was very sensitive to MMS.Furthermore,using comet technique and staining with propidium iodide(PI),the results showed that the DNA fragments in the mre11 were far more than those in wild type,indicating that OsMre11 participated in the DNA damage repair process.6.OsMre11 interacted directly with OsRad50 and OsNbs1 to form a complex.The three-dimensional modeling of Mre11,Rad50 and Nbs1 proteins in different species showed that they were highly similar in structures,indicating that their proteins may have a certain degree of conservation in the high-level structures.Using Bi FC combined with Co-IP technique to analyze the interaction relationship among OsMre11,OsRad50 and OsNbs1,the results showed that OsMre11 could interact directly with OsRad50 and OsNbs1 respectively,indicating that OsMre11 could be used as an intermediate bridge connecting OsRad50 and OsNbs1 to form an MRN complex,playing a role in the rice growth and development together.