高原鼠兔诱导型一氧化氮合酶cDNA克隆及序列分析  

Cloning and Characterization of Inducible Nitric Oxide Synthase from Plateau Pika(Ochotona curzoniae)

作　　者：谢玲[1,2] 郭新异[1,2] 张湑泽 庞礴[1,2] 都玉蓉[4] 邹小艳[1] 郭松长[1] 赵新全[1] 

机构地区：[1]中国科学院西北高原生物研究所,中国科学院高原生物适应与进化重点实验室,西宁810008 [2]中国科学院大学,北京100049 [3]青海民族大学,西宁810007 [4]青海师范大学生命与地理科学学院,西宁810008

出　　处：《四川动物》2014年第4期511-521,共11页Sichuan Journal of Zoology

基　　金：国家自然科学基金青年科学基金资助项目(30800118);中国科学院知识创新工程重要方向项目(KSCX2-EW-J-26);青海省科技计划类项目(2013-Z-750)

摘　　要：高原鼠兔Ochotona curzoniae，世居在青藏高原海拔3000-5000 m的地区，是一种典型的低氧耐受哺乳动物。一氧化氮（NO）作为一种有效的血管舒张因子，在预防低氧诱导的低氧性肺血管收缩反应和肺动脉高压中发挥着重要功能。诱导型一氧化氮合酶（iNOS）是一种催化L-精氨酸合成NO的重要酶，受低氧调控。本研究经RT-PCR和cDNA3’末端快速扩增（3’RACE）方法成功克隆了高原鼠兔iNOS基因cDNA序列，并对其分子特征进行了分析。结果显示：高原鼠兔iNOS cDNA全长为3981 bp，开放阅读框（ORF）为3450 bp，共编码1149个氨基酸残基；预测的蛋白序列与北美鼠兔、兔、人、大鼠、小鼠、狗以及猪的同源性分别为98%、87%、82%、78%、78%、82%和83%；蛋白结构预测结果显示高原鼠兔iNOS具有氧化域、还原域及黄素腺苷酸结合区域等iNOS所具有的典型结构域；基于iNOS的最大似然树和贝叶斯树均支持鼠兔与兔有最近的亲缘关系，与形态或其他分子标记构建的进化关系相符；分子进化分析检测到高原鼠兔iNOS中存在3个正选择位点——32T、33Y和46R，但不同模型的结果表明哺乳动物iNOS基因所受选择以净化选择为主，不支持iNOS在高原鼠兔支系发生适应性进化。该研究为揭示高原鼠兔iNOS的表达特征及其在低氧适应中的作用与调控机制研究奠定了初步基础。Plateau pika （Ochotona curzoniae）, a native species living at 3000-5000 mabove sea-level on the Qinghai-Tibetan Plateau, is a typical hypoxia-tolerant mammal. Nitric oxide （NO）, which has been proved to be a vasodilator, plays important roles in preventing hypoxia-induced hypoxic pulmonary vasoconstriction and pulmonary hypertension under hypoxia. Inducible nitric oxide synthase （iNOS） is a key enzymes that catalyzing L-arginine to NO and regulated by hypoxia stress. In this study, the cDNA of iNOS was cloned by RT-PCR and3’-rapid amplification PCR of cDNA ends （3’-RACE） from the lung of plateau pika. The results showed that the length of iNOS cDNA of plateau pika was 3981 bp, containing an open reading frame （ORF） of 3450 bp and encoding 1149 amino acid residues. The deduced amino acid sequence was highly identical to those of the American pika （98%）, followed by rabbit （87%）, human （82%）, rat （78%）, mouse （78%）, dog （82%） and pig （83%）. The typical iNOS domains, including oxidase domain, reductase domain and FAD binding site, were identified in the iNOS of plateau pika. The molecular phylogenetic trees based on ML and Bayesian methods showed that pika has a close relationship with rabbit. Three positive selection sites, 32T, 33Y and 46R, were identified by the PAML analysis. However, the modeling results showed that the evolution of iNOS was mainly under negative selection, suggested that there was no adaptive evolution in plateau pika iNOS. This study laid a foundation for further exploration on the expression pattern, function and regulatory mechanism of iNOS in plateau pika adapting to high altitude environment.

关 键 词：高原鼠兔 诱导型一氧化氮合酶 CDNA克隆 

分 类 号：Q959.8[生物学—动物学] Q78