Cloning And Expression Regulation Of DNA Glycosylase Gene From Antarctic Ice Algae Chlamydomonas Sp. ICE-L

DNA repair is the main factor to maintain the genetic stability of species DNA,which is of great significance to human health and disease treatment.The identification of E.coli uracil DNA glycosylase marks the discovery of base excision repair(BER).The current view is that BER plays an important role in cancer,neurodegeneration and aging.BER is initiated by DNA glycosylase,which can recognize specific damaged bases in DNA and remove them.Then,endonuclease,repair polymerase and ligase were used to further treat the resulting base free damage,and finally the original DNA sequence was completely restored.Antarctic ice algae grow in the Antarctic sea ice environment with low temperature,high salt and strong ultraviolet radiation,and their DNA is very vulnerable to damage.DNA damage repair is very efficient.This study is the first to carry out the cloning,expression and enzymatic properties of DNA glycosylase gene of Antarctic ice algae,in order to explore the specific molecular mechanism of DNA glycosylase of Antarctic ice algae.Objective: In this study,Antarctic ice alga Chlamydomonas sp.ICE-L,which can thrive in the extreme sea ice habitat,was used as the research object.The cloning and expression regulation of its DNA glycosylase gene were studied to clarify the expression pattern of DNA glycosylase of Antarctic ice alga in its extreme environments such as strong ultraviolet and high salinity.And the specific molecular mechanism of DNA glycosylase of Antarctic ice alga was explored in the extreme sea ice environment.Methods:(1)DNA glycosylase gene sequence information was screened according to the whole gene sequencing results of Antarctic ice algae C.sp.ICE-L,primers were designed,DNA glycosylase gene of Antarctic ice algae was cloned,and bioinformatics analysis was conducted.(2)The regulation of DNA glycosylase gene expression in Antarctic ice algae in extreme low temperature,high salinity and strong ultraviolet environment was analyzed by real-time fluorescent quantitative PCR.(3)The expression vector of uracil DNA glycosylase from Antarctic ice algae was constructed,and the recombinant Pichia pastoris engineering strain was obtainedand heterologously expressed.The recombinant uracil DNA glycosylase from Pichia pastoris was isolated and purified,and its activity was detected by using a double chain probe method.Results:(1)Formamidopyrimidine-DNA glycosylase gene Ci FPG1,Adenine-DNA glycosylase gene Ci ADG1,and Uracil-DNA glycosylase gene Ci UDG1 of C.sp.ICE-L were obtained by PCR.The DNA glycosylases Ci FPG1,Ci ADG1,and Ci UDG1 of Antarctic ice algae all contain DNA binding functional domains and conserved regions;According to phylogenetic tree analysis,the evolutionary relationship between Antarctic ice algae Ci FPG1 and mosses and higher plants is relatively close,while the evolutionary relationship between Ci ADG1 and Ci UDG1 and algae is relatively close.(2)The transcriptional changes of DNA glycosylase genes Ci FPG1,Ci ADG1,and Ci UDG1 in Antarctic ice algae were significant under low temperature treatment and optimal temperature,and the expression level increased under extreme low temperature environment,while the expression level did not change significantly under high temperature treatment.The expression level of Ci ADG1 was the highest at the optimal salinity of 32 ‰,Ci UDG1 was the highest at the salinity of 96 ‰,and Ci FPG1 was the highest at the salinity of 128 ‰.The DNA glycosylase gene Ci FPG1,Ci ADG1,and Ci UDG1 of Antarctic ice algae can quickly respond to low intensity UVB,and their expression levels were increased under low intensity UVB stress,while the expression level of the gene is the lowest under high intensity UVB stress.(3)The fusion expression vector p PICZ A-Ci UDG1 of the uracil DNA glycosylase Ci UDG1 was constructed from Antarctic ice algae and transfer it to Pichia pastoris receptive cells.The positive clones was screened,and induced to express to obtain a protein with a molecular weight of 60 KD;The cultivation of recombinant engineering bacteria were expanded,which were separated and purified by using Flag label affinity chromatography and nickel column affinity chromatography,respectively.It was found that using nickel column affinity chromatography,with using a chromatography buffer of(PBS+8M Urea),can achieve good separation and purification effects,which showed the electrophoretic bands of the target protein was uniformed,and successfully purified Antarctic ice algae Ci UDG1.After purification,the Ci UDG1 enzyme concentration of Antarctic ice algae was 0.24 mg/m L,the specific activity was 2047.6 U/mg,and the thermal stability was low.Conclusions: In this study,DNA glycosylase genes Ci FPG1,Ci ADG1,and Ci UDG1 were cloned from Antarctic ice algae C.sp.ICE-L,and their bioinformatics analysis was completed.The variation of DNA glycosylase gene expression in Antarctic ice algae under different temperature and salinity treatments may be related to the adaptation of Antarctic ice algae to extreme sea ice salt sac environments.The DNA glycosylase gene of Antarctic ice algae can quickly respond to low intensity UVB to adapt to the strong ultraviolet radiation environment in Antarctica.Recombinant Pichia pastoris engineering strain of uracil DNA glycosylase were constructed and heterologously expressed.The recombinant uracil DNA glycosylase from Pichia pastoris was isolated and purified.It was found that the uracil DNA glycosylase has good enzymatic activity,which will lay a solid foundation for the development and utilization of DNA glycosylase from Antarctic ice algae.