Cloning And Function Analysis Of DNA Damage Repairing Response Pathway Related Genes In Halostachys Caspica Under Salt Stress

DNA is the main genetic material,which plays an important role in the transmission and transmission of genetic information.However,a variety of abiotic and biotic stresses,such as salt,ultraviolet and ionizing radiation,chemical mutagens,free radicals,bacteria and viruses and so on,can lead to DNA damage,which may lead to changes in the physical and chemical properties of DNA,resulting in cellular and genetic toxicity.It mainly includes oxidative damage,DNA single strand breaks,DNA double strand breaks and so on.Plants,with their inherent immobility and obligatory dependence on sunlight for energy,face tremendous challenges in maintaining the integrity of the genome,which is under continuous assault from environmental factors like UV and ionizing radiation,high salinity,chemical mutagens,and free radicals or alkylating agents generated by endogenous processes.Therefore,plant cells have evolved with highly efficient and wide-ranging mechanisms for the detection and repair of DNA damage to ensure genome stability.DNA double-strand breaks(DSBs)is one of the most toxic forms of DNA damage induced by all kinds of stress.There are two main ways of DSBs repair: non homologous end joining(NHEJ)and homologous recombination repair(HR).Halostachys caspica is an Amaranthaceae perennial shrub,is a typical salt tolerant plants in Xinjiang desert area.With its unique environmental adaptability,it has become an ideal material for salt tolerance research.Our research group previous study on transcriptome sequencing analysis of Halostachys caspicaan under salt stress,and found that a class unigene which related of DNA damage repair was up-regulated.In this study,2 genes which related to DSBs repair were selected from the UniGene associated with DNA damage repair,HcDNA polymerase lambda(HcDNApol?)and DNA meiotic recombinase 1(HcDMC1).DNApol lambda participates in the repair of DSBs through non homologous terminal pathway,and DMC1 participates in homologous recombination repair pathway to repair DSBs.The full-length of the gene was cloned by RACE,and its tolerance under salt stress was analyzed by prokaryotic and eukaryotic yeast system to study their role in DSBs repair,which laid the foundation for the further study on salt tolerant mechanism of salt spike.The main results are as followed.1.According to the transcriptome of Halostachys caspica under salt stress,a DNA damage repair gene DNA polymerase lambda(HcDNApol?)was cloned from Halostachys caspica by RACE.Sequence analysis indicated that HcDNApol? contains an open reading frame of 1335 bp,which encodes 444 amino acids.Conserved domain analysis showed that HcDNApol? was the number of DNA polymerase X family,and phylogenetic tree analysis indicated that HcDNApol? was an independent branch.Real time quantitative PCR results showed that the expression of HcDNApol? gene after gradually increasing NaCl salt stress was rapidly up-regulated and reached the highest level on 14 days.The expression of HcDNApol? gene could be induced by salt stress.In order to study on the salt tolerance of HcDNApol?.In this study,the prokaryotic expression vector was constructed,the His-HcDNApol? protein was purified,and the antibody of HcDMC1 was obtained.The growth curves of recombinant bacteria were detected under different concentrations of NaCl stress.The results showed that overexpression of HcDNApol? in E.coli cells could increase the tolerance to salt stress.2.Prokaryotic expression and salt tolerance analysis of Halostachys caspica HcDMC1.Based on the previous cloned DNA repaired gene from Halostachys caspica,and the expression level of salt stress,showed that HcDMC1 could be induced by salt stress.In order to study on the salt tolerance of HcDMC1.In this study,the prokaryotic expression vector was constructed,the HcDMC1 protein was purified,and the antibody of HcDMC1 was obtained.The growth curves of recombinant bacteria were detected under different concentrations of NaCl stress.The eukaryotic expression vector was constructed,and the growth status of recombinant yeast was detected under different concentrations of NaCl stress.The results showed that overexpression of HcDMC1 in E.coli and yeast cells could increase the salt tolerance.The above results indicate that HcDNApol? and HcDMC1 participate in the plant response to salt stress,can improve the stress of recombinant bacteria growth.