高原鼢鼠和高原鼠兔肺组织磷脂酰胆碱合成关键酶基因的进化及其表达差异分析

Evolution Analysis and Expression Difference of Key Enzyme Genes for Phosphatidylcholine Synthesis in Lung Tissues Among Plateau Zokors and Plateau Pikas

肺表面活性物质有助于维持肺泡结构,促进呼吸作用和对氧的吸收及利用,磷脂酰胆碱为肺表面活性物质磷脂主要成分。为进一步研究高原动物对低氧环境的适应机制,本文以青藏高原特有物种高原鼢鼠(Eospalax baileyi)和高原鼠兔(Ochotona curzoniae)为研究对象,应用生物信息学方法对编码磷脂酰胆碱合成途径中关键酶胆碱激酶基因Chok-α和Chok-β、磷酸胆碱胞苷转移酶基因Pcyt-α和Pcyt-β以及胆碱磷酸转移酶基因Cpt的序列进行进化分析,并以SD大鼠(Rattus norvegicus)为对照,测定了这些基因在肺组织中的mRNA表达水平。生物信息学结果表明,Chok-α、Chok-β、Pcyt-α、Pcyt-β和Cpt基因序列高原鼢鼠与以色列鼹鼠(Nannospalax galili)的同源性最高,高原鼠兔与北美鼠兔(O.princeps)的同源性最高,均高达90%以上;高原鼢鼠Cpt与以色列鼹鼠,高原鼠兔Chok-β、Pcyt-β和Cpt与北美鼠兔有平行进化位点。选择压力分析表明,高原鼢鼠Chok-α亚基第4位的赖氨酸、第5位点的苯丙氨酸和第10位的谷氨酸,高原鼠兔Chok-β亚基第4位蛋氨酸,高原鼢鼠Cpt第163位谷氨酸,这些位点均存在显著差异(P<0.05);SIFT评估结果发现,高原鼠兔的Chok-β亚基中第212位氨基酸变异位点和Pcyt-β亚基第18位氨基酸变异位点对其功能有显著影响(P<0.05)。mRNA表达水平分析结果表明,高原鼢鼠Chok-α和Chok-βmRNA表达水平均显著高于高原鼠兔与SD大鼠(P<0.01),高原鼠兔Chok-βmRNA表达水平显著高于SD大鼠(P<0.05);SD大鼠Pcyt-α、Pcyt-β和Cpt mRNA表达水平显著高于高原鼠兔和高原鼢鼠(P<0.01),而高原鼠兔与高原鼢鼠间无差异(P>0.05)。以上结果表明,与SD大鼠相比,高原鼢鼠和高原鼠兔磷脂酰胆碱合成途径中关键酶氨基酸变异和基因表达水平的差异以及两种高原动物的生理适应,更利于它们在高寒低氧的独特环境中获取氧并利用氧,从而促进呼吸作用,以加强能量代谢并适应低氧环境。

[Objectives]Pulmonary surfactant(PS)can help to maintain the structure of alveoli,improve respiration and oxygen absorption and utilization.Notably,phosphatidylcholine(PC)serves as the principal constituent of pulmonary surfactant phospholipids.In order to shed lights into the adaptation mechanism of plateau animals to hypoxic environments,this study focuses on the endemic species of Plateau Zokor Eospalax baileyi and Plateau Pika Ochotona curzoniae in the Qinghai Tibet Plateau.[Methods]The sequences of Chok-α,Chok-β,Pcyt-α,Pcyt-βand Cpt genes were analyzed by MEGA 7.0,PAML 4.8 program and Ancestor program.Additionally,the mRNA expression levels of these genes were determined with Real-time PCR in the lung tissues and compared with those in the SD Rat Rattus norvegicus.[Results]The bioinformatics analysis revealed that the sequences of Chok-α,Chok-β,Pcyt-α,Pcyt-βand Cpt in E.baileyi were highly homologous with those of Nannospalax galili,as well as the O.curzoniae has the highest level of homology with O.princeps(>90%)(Table 2).Cpt of E.baileyi and N.galili,Chok-β,Pcyt-βand Cpt of O.curzoniae and O.princeps occurred convergent sites.Selection pressure analyses showed that lysine at sites 4,phenylalanine at sites 5 and glutamic acid at sites 10 of Chok-αsubunit in Plateau Zokor were significantly different(P<0.05,Table 3);methionine at sites 4 of Chok-βsubunit in Plateau Pika,glutamic acid at sites 163 of Cpt subunit in Plateau Zokor were also significantly different(P<0.05,Table 3).SIFT test showed that No.212 variation sites of Chok-βsubunit and No.18 variation sites of Pcyt-βsubunit in Plateau Pika had significant effects on gene function(P<0.05,Table 4).The mRNA expression levels of Chok-α,Chok-β,Pcyt-αand Pcyt-βin Plateau Zokor were significantly higher than those in Plateau Pika and SD Rat(P<0.01),and the mRNA expression level of Chok-βin Plateau Pika was higher than SD Rat(P<0.05)(Fig.6).The expression level of Cpt in SD Rat was significantly higher than that in Plateau Pika and Plateau Zokor(P<0.01),while there was no difference between expression levels in Plateau Pika and Plateau Zokor(P>0.05)(Fig.6).[Conclusion]The above results demonstrate that,in comparison to SD Rat,the amino acid structure and gene expression variations of key enzyme genes in phosphatidylcholine pathway of Plateau Zokor and Plateau Pika,along with two plateau animal physiological adaptation make them more conducive to acquire and employ oxygen in the unique environment of high cold and hypoxic environment.Consequently,this reinforces respiration,enhances energy metabolism,and facilitates adaption to low oxygen environments.