猪獾苦味受体T2R2基因的分子克隆与进化分析.pdfVIP

猪獾苦味受体 T2R2 基因的分子克隆与进化分析 徐怀亮 ， 姚永芳， 朱庆∗ 四川农业大学动物科技学院，雅安 625014 摘要：苦味的感知是机体有效的自我保护机制之一。文章采用PCR和克隆测序方法首次从猪獾基因组中获得一全长为1 169 bp 的苦味受体T2R2基因DNA序列（GenBank登录号: FJ812727）。该序列含有完整的1个外显子（无内含子），大小为915 bp，编 码304个氨基酸残基。其蛋白质等电点为9.76，分子量为34.74 kDa 。拓扑结构预测显示猪獾T2R2蛋白上含有N−糖基化位点、 N- 肉豆蔻酰化位点各1个，蛋白激酶C磷酸化位点2个。整个蛋白质多肽链含有7个跨膜螺旋区，4个细胞外区和4个细胞内区。 亲水性/疏水性分析表明，猪獾T2R2蛋白质为一疏水性蛋白，其亲水性区段所占比例较小。种间相似性比较显示，猪獾T2R2 基因与犬、猫、牛、马、黑猩猩和小鼠的T2R2基因cDNA序列相似性分别为91.4%、90.6%、84.4%、85.4%、83.8%、72.1%， 氨基酸序列相似性分别为85.5%、85.8%、74.0%、77.6%、75.3%、61.5% 。核苷酸替换计算和选择性检验结果表明，猪獾T2R2 基因与犬、猫、牛、马、黑猩猩和小鼠间存在着强烈的纯净化选择(Purifying selection) ，即强烈的功能束缚（Functional constraint ），进一步分析发现该选择作用实际上主要存在于跨膜区。猪獾、犬、猫、牛、马、黑猩猩和小鼠的T2R2基因外显 子核苷酸序列构建的基因树与其物种树的拓扑结构是相一致的，表明T2R2基因适合于构建不同物种间的系统进化树。 关键词：猪獾；苦味受体；T2R2基因；克隆；分子进化分析 Molecular cloning and evolutionary analysis of Hog-Badger bitter taste receptor T2R2 gene ∗ ∗ XU Huai-Liang , YAO Yong-Fang, ZHU Qing College of Animal Science and Technology, Sichuan Agricultural University, Ya’an 625014, China Abstract: Recognition of natural bitter toxins through taste is one of the most effective mechanisms of self-safety. An approximate 1 169 bp sequence of the bitter taste receptor T2R2 gene was obtained by PCR and cloning technique from hog-badger genomic DNA （GenBank accession number : FJ812727 ）. This sequence contains a complete single exon (without intron) 915 bp in size, which encodes 304 amino acid residues. The isoelectric point （pI ） of the protein is 9.76 and its molecular weight is 34.74 kDa. Topology prediction showed that the T2R2 protein contained one N-glycosylation site, one N-myristoylation site, and two potential protein kinase C phosphorylation sites. Additionally, the whole peptide chains were comprised of seven transmembrane helix regions, four extracellular regions, and four intracellular regions. The T2R2 is a hydrophobic protein with a few hydrophilic co