猪獾苦味受体T2R2基因的分子克隆与进化分析

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。

Molecular cloning and evolutionary analysis of hog badger bitter taste receptor T2R2 gene

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 mo-lecular 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 chain was comprised of seven transmembrane helix regions, four extracellular regions, and four intracellular regions. The T2R2 is a hydrophobic protein with a few hydrophilic components. Homology analysis of the T2R2 gene sequences by Clustal Windicated that the cDNA sequence homology of T2R2 gene in hog badger with dog, cat, cattle, horse, chimpanzee, and mouse is 91.4%, 90.6%, 84.4%, 85.4%, 83.8%and 72.1%, respectively, and the homology of amino acid sequence is 85.5%, 85.8%, 74.0%, 77.6%, 75.3% and 61.5%, respectively. The results of nucleotide acid substitution computation and selective test showed that strong purifying selection (functional constraint) occurred between hog badger and the six species, respec-tively, which mainly existed in the transmembrane regions of T2R2. In addition, the Neighbour-Joining tree of T2R2 gene exons from these seven species is consistent with their species tree, indicating that the T2R2 gene is suitable for constructing molecular phylogenetic tree among different species likewise.