The Response Of Plateau Zokor P53 To Hypoxia And The Regulation Specificity To The Expression Of The Target Genes

The plateau zokor,Myospalax baileyi,is a specialized subterranean rodent living on the Qinghai-Tibet Plateau,and the inhabits sealed burrows at an altitude of 2 800 m to 4 200 m.p53,a tumor suppressor gene that plays a crucial role in cell cycle arrest,apoptotic and DNA damage repair and angiogenesis by regulating its target genes,and have an important role in animals' longevity and anti-cancer.In order to shed lights into the adaptation mechanisms to the hypoxic burrowing environments in the plateau zokor,we cloned the p53 coding DNA sequences,the homologies,positive selection sites and the parallel evolution sites of p53 and target genes were analyzed by MEGA 7.0,PAML4.8 program and Ancestor program,respectively.In addition,the expression levels of these genes were measured by qPCR in plateau zokor tissues under different altitudes(3 300 m and 2 260 m)and compared with that of SD rat in this study.Our results showed that,(1)the coding DNA sequence is 1179 bp,consisting of 392 amino acid residues.The nucleotide and amino acid sequences of the plateau zokor p53 had highly homologies with Eospalax cansus and Nannospalax galili,and the homologies were 98.47%,98.98% and 93.30%,95.41%,respectively.Compared to Eospalax cansus,Nannospalax galili,Heterocephalus glaber and Rattus norvegicus p53,plateau zokor had 4,17,48 and 59 mutation sites,and had 2,3,12 and 16 mutation sites within the DNA-binding domain,respectively.Compared to human p53,the subterranean rodents p53 has two mutation sites in common with the human hotspots in the DNA-binding domain at 246(248 in human)and 271(273 in human).Compared to subterranean rodents,plateau zokor have a mutation at residue 309.(2)The expression levels of p53 in plateau zokor tissues increased significantly from 2 260 m to 3 300 m(P<0.01),but there was no significant difference in rat at those altitudes(P>0.05).(3)the nucleotide and amino acid sequences of the cell cycle,apoptosis and DNA damage repair target genes in plateau zokor had highly homologies with Nannospalax galili,and the homologies were exceeded 90%;p21,CyclinD1,CyclinE,CyclinB1,PIDD,PUMA,Apaf-1,IGFBP3 Bcl-2,p48 and p53R2 of plateau zokor and Nannospalax galili occurred parallel sites.The SIFT test showed that 27,105,853,157,320 and 285 variation sites had effects on the function of p21,CyclinB1,PIDD,PUMA,Apaf-1 and IGFBP3,respectively.(4)compared to low altitude(2 260 m),the expression level of p21 was significant increased in plateau zokor of liver,lung and skeletal muscle tissues(P<0.01),and the p21 downstream genes had tissue specific,CyclinD1,CyclinE,CDK6 and CDK2 were significant decreased in liver tissues(P<0.05),CyclinD1 and CDK6 were significant decreased in lung(P<0.05),whereas the levels of these genes were no significant difference in skeletal muscle(P>0.05).The levels of Gadd45?,B99,14-3-3-? and CyclinB1 were no significant difference in plateau zokor tissues under different altitudes(P>0.05),and the levels of these genes were no significant difference in SD rat(P>0.05).(5)under high altitude(3 300 m),the expression levels of proapoptotic genes Bax,PUMA and Apaf-1 were significant decreased in liver(P<0.05),PIDD,Bax,PUMA and Apaf-1 were significant decreased in lung(P<0.05),Fas,Bax,PUMA and Apaf-1 were significant decreased in skeletal muscle of plateau zokor(P<0.01).Under high altitude,the levels of antiapoptotic genes of Bcl-2 and the ratio of Bcl-2/Bax was prominently increased in liver,lung and skeletal muscle tissues(P<0.01),whereas the SD rat was not(P>0.05).(6)compared to the low altitude(2 260 m),the levels of p48 and p53R2 were significant increased under high altitude(3 300 m)in plateau zokor of liver,lung and skeletal muscle tissues(P<0.05)whereas SD rat was not(P>0.05).The results indicated that,plateau zokor p53 had common variation sites and different variation sites as well,the further the phylogenetic relationships of species,the greater the variation of the p53;the greater difference between plateau zokor and rat,which indicated that the living environment has a great influence on the variation of p53.p53 is sensitive to the oxygen concentration in plateau zokor,and hypoxia upregulates the levels of p53,whereas SD rat was not.For the long-time adaptation to the hypoxic environments,p21 was up-regulated and inhibit the levels of target genes in plateau zokor tissues,causing the cell cycle G1 arrest,which provide sufficient time for DNA repair,and hypoxia up-regulate the levels of p48 and p53R2 to repair the damaged DNA and improve the DNA repair function,and ensure the accuracy of DNA replication,which was the common characteristics of the anti-cancer in subterrean rodents;the expression levels of proapoptotic genes were decreased,and the level of antiapoptotic gene was increased,so as to inhibit apoptosis under hypoxic environments,it ensure the life span of the cells,which may related to the longevity of the subterrean rodents.At the same time,the regulation of the cell cycle and apoptosis in plateau zokor was not only related to the expression levels of the genes,but also may be related to the structural variation of p21,CyclinB1,PIDD,PUMA,Apaf-1 and IGFBP3,which is one of the molecular mechanisms of plateau zokor to adapt to the hypoxic-hypercapnic burrowing environments.