Expression And Function Of CPD Photolyase PnPHR1 From Pohlia Nutans

DNA,as the carrier of biological genetic material,is a biological macromolecule necessary for the development and normal operation of organisms.Ultraviolet B(UV-B,280nm-320nm)can induce the adjacent pyrimidine bases on the single strand of DNA to form covalent bonds and produce pyrimidine dimers.Pyrimidine dimers hinders the replication and transcription of DNA and affects the functions of proteins and eventually leads to the change of genetic material.Photolyase is an enzyme responsible for DNAUV-B damage repair.It can specifically and effectively break the covalent bond between pyrimidine dimers,restore them to the monomer structure,and restore the normal state of DNA.At present,photolyase has been found in bacteria,fungi,algae,higher plants and some vertebrates.But no photolyase has been found in human body.If the UV-B damage of human skin cells cannot be repaired in time,a variety of problems will occur,such as skin inflammation,immunosuppression,accelerated aging and even cancer.The Antarctica is one of the areas with the strongest UV-B radiation,posing a severe challenge to the survival of living organisms.Moss is the most widely distributed and abundant plant on the continent.In this paper,PnPHR1,a CPD photolyase gene with a strong response to UV-B radiation,was selected from Pohlia nutans and the functions were studied.The specific experimental results are as follows:1.Bioinformatics analysis of the CPD photolyase from Pohlia nutansA highly responsive gene was cloned and expressed from the transcriptome of Pohlia nutans under UV-B stress.Sequence analysis showed that the full length of the gene was 1830bp,encoding a protein with 609 amino acids and a molecular weight of 69.1KDa.It was preliminarily identified as the type ? CPD photolyase and named as PnPHR1.We found that PnPHR1 belongs to the photolyase/cryptochrome family,and shared 68.0%similarity with Physcomitrella patens by multi-sequence analysis.Phylogenetic analysis showed that(6-4)PPs and cryptochrome group were clusterd together and CPD photolyase was evolved into another branch.And PnPHR1 was clustered with the the photolyase of P.patens.The three-dimensional structure simulation showed that the N-terminal of PnPHR1 was an ?/? domain,and the C-terminal wes a regions.The catalytic auxiliary group FAD was located in the C-terminal structure and bound to the damaged DNA molecule through electrostatic action and hydrophobic action.2.Activity detection of Pohlia nutans CPD photolyaseThe expression vector PnPHRl-pGEX-4T-1 was constructed and the protein was expressed in E.coli.Synthetic Oligo(dT)16 was treated with UV-B to form CPD oligonucleotides(OD260 decreased from 1.6 to 0.36).CPD oligonucleotide was used as the reaction substrate and PnPHRl protein was added to analyze the activity in vitro.After 4h,the reaction system OD260 was increased by 0.22,while the absorbance of the control system remained unchanged.The results demonstrated that PnPHRl could repair the CPD oligonucleotide,break the covalent bond between its adjacent thymine and restore the pyrimidine monomer structure.Subsequently,the reaction conditions of PnPHR1 were explored,and it was found that the optimal reaction conditions were 20? and pH8.5.SY2 is an E.coli.CPD photolyase defective strain,which is sensitive to UV-B.PnPHRl increased the survival rate of SY2 after UV-B irradiation from 4.4%to 17.4%.HaCaT are human keratinocytes cells,which was cultured for 24 hours after UV-B radiation.Compared with the control group,HaCaT cells incubated with PnPHR1 had fewer fragments,clear cell membrane boundaries,with a higher survival rate.Thus,PnPHR1 has the activity of photolyase and can repair the damage of UV-B to HaCaT cells.3.Study on the function of PnPHR1 in abiotic stressThe recombinant vector PnPHR1-pRI101 was introduced into Arabidopsis by agrobacterium transformation to obtain overexpressed PnPHR1 Arabidopsis.Then we analyzed the effect of PnPHR1 on abiotic stresses such as UV-B,H2O2 and NaCl.Under normal conditions,Arabidopsis grows well,with green leaves,round and saturated shape,abundant stem and large leaf surface area.There are no difference between wild type and overexpressed PnPHR1 Arabidopsis.After 12h of UV-B irradiation,the growth of wild type was significantly inhibited.The stem was fine,the leaves were yellow and atrophied of wild type Arabidopsis.Compared with the wild type,the overexpression Arabidopsis had longer,greenish leaves and larger leaf surface area.DAB staining showed that the AtOEs after UV-B stress had lower degree of oxidative damage in vivo.qRT-PCR results showed that the expression levels of related genes(AtCOP1 and AtUVR2)and antioxidant enzyme genes(AtCAT1 and AtFe-SOD1)in transgenic Arabidopsis were significantly increased after UV-B treatment.These results indicated that PnPHRl reduced the production of reactive oxygen species(ROS)by enhancing the signal transmission of UV-B and the activity of CAT and SOD,improved the tolerance to UV-B radiation in plants.In addition,under NaC1 stress,the root length of overexpressed lines was significantly longer than that of the wild type,and the relative expression levels of ion-regulated transporter genes AtNHX1,AtHKT1 and AtP5CS were up-regulated.It showed that PnPHR1 enhanced the resistance to NaCl stress in Arabidopsis.Both UV-B and salt can cause oxidative stress.The effect of PnPHRl on oxidative stress was further studied.Under 0.75mM H2O2 treatment,the average root length of WT was 1.48cm,while the root length of AtOEl and AtOE2 was 2.35cm and 2.26cm,respectively.Under the treatment of ImM H2O2,the root lengths of AtOEl and AtOE2 were 1.73 and 1.75 times comparison with WT,respectively.qRT-PCR results showed that PnPHRl reduced the expression of AtRbohC and AtRbohE.In summary,PnPHRl can repair CPD oligonucleotides,improve the survival rate of SY2 and HaCaT after UV-B irradiation.Overexpression of PnPHR1enhanced its resistance to abiotic stresses such as UV-B,NaCl and H2O2 by regulating the expression of antioxidant enzymes and ion transporters.This experiment provides an important reference for the study of moss adaptation to the polar environment,enriches the genetic resources of Antarctic plants,and lays a foundation for the industrial application of this gene.