Functional Study Of The RecQ Family Belonging To Dna Helicase In Rice

DNA helicase is a motor protein that uses the energy of ATP hydrolysis to catalyze the unwinding of double-stranded DNA.It is essential for the most basic processes of genetic information replication,transcription,repair and recombination.Therefore,DNA helicase is highly conserved among organisms.Rec Q helicase is an important member of DNA helicase SFⅡ.The first Rec Q helicase was discovered in E.coli during the study of the mechanism of thymine defect death.Subsequently,Rec Q helicase was also found in eukaryotic cells.Rec Q family contains six subfamilies,from Rec Q1 to Rec Q6 subfamilies.They also play important roles in eukaryotic DNA replication,repair,telomere maintenance and homologous recombination.There are seven Os Rec Q genes in rice.At present,many studies focus on the function of Os Rec Q4protein,which was found to be involved in DNA repair and homologous recombination,and few reports about the functions of other Os Rec Q genes.In recent years,clustered regularly interspersed short palindromic repeats/CRISPR-associated protein 9（CRISPR/Cas9）technology is developing rapidly.We could use CRISPR/Cas9 technology to knockout candidate genes and get its mutants fast and effective,thus many scientists use reverse genetics to study gene function.In this study,we used CRISPR/Cas9 technology to knockout of Os Rec Q gene family in rice respectively to obtain seven Os Rec Q gene mutants,and observe their phenotype and cytology.The purpose of our study is to preliminarily understand the role of each Os Rec Q protein during rice development and analyze its biological function.Due to understanding Os Rec Q helicase biological function and mechanism give a way to use them to improve gene recombination frequency.This will provide clues for improving the efficiency of rice hybrid breeding and developing new tools for gene targeting in plant.First,we designed the knockout target sequence of seven Os Rec Q gene and constructed the knockout vector respectively.Then,we transformed rice callus by agrobacterium-mediated method and screening the positive transgenic plants.It was found that we had obtained homozygous genetic material with a large number of mutants,including three homozygous mutants of osrecq1,osrecq2 and osrecq7,four of osrecq3,two of osrecq4 and osrecq5,and five of osrecq6.There were no significant differences in plant height,tiller number,leaf size and panicle type between each mutant and the wild type.We found that the overall seed setting rate of osrecq1 and osrecq2 mutants was about 70%,osrecq3 is only more than30%,osrecq4 and osrecq7 was about 50%,osrecq6 is above 70%.In conclusion,the fertility of the osrecq mutant is lower than that of the wild type,suggesting that Os Rec Q protein may be involved in the reproductive process of rice in various degrees.The meiosis process of each mutant was further observed.At present,we haven’t found abnormalities of osrecq1,osrecq2,osrecq3 and osrecq7 mutants.However,the number of chromosomes in osrecq4 mutants were abnormal during Diakinesis and the number and morphology of chromosomes in osrecq6 mutants are abnormal during metaphaseⅠand telophaseⅡ.Therefore,we speculate that Os Rec Q4 and Os Rec Q6 may involve in homologous recombination during meiosis.osrecq6 and osrecq7 mutants were treated with different concentrations of H₂O₂which belongs to the DNA damage agent.After that,we observed seedling leaves,plant height and root condition.We found osrecq6 and osrecq7 showed different sensitivity to hydrogen peroxide treatment.The result indicate that Os Rec Q6 inhibits the repair process of DNA oxidative damage caused by hydrogen peroxide,while Os Rec Q7promotes the repair process after DNA oxidative damage.We have the T₁ and T₂ genetic materials of osrecq6 mutant,while the rest of the mutants are T₀ genetic materials.Therefore,we can’t exclude the phenotypic inconsistencies of each gene mutant are due to plant culture or rice culture conditions.Furthermore,the major challenge in studying Rec Q proteins of plants is to elucidate their respective roles both in recombination and repair pathways.In summary,the conclusions of this study are as follows.Firstly,we obtained different osrecq mutant,providing materials for subsequent gene function exploration.Secondly,Os Rec Q has different degrees of impact on Oryza sativa fertility.Thirdly,Os Rec Q4 and Os Rec Q6 proteins may affect the process of homologous recombination.Fourthly,Os Rec Q6 and Os Rec Q7 participate in DNA repair process,but they have different effects.