黑熊苦味受体Tas2R2基因的分子克隆与序列分析

Molecular Cloning and Sequence Analysis of Black Bear(Ursus thibetanus) Bitter Taste Receptor Tas2R2 Gene

实验采用PCR和克隆测序方法首次从黑熊基因组中获得一全长为1 019 bp的苦味受体Tas2R2基因DNA序列(GenBank登录号:JQ239035)。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9.49,分子量为34.80 kDa。拓扑结构预测显示黑熊Tas2R2蛋白上含有3个N糖基化位点、4个丝氨酸磷酸化位点、3个色氨酸磷酸化位点和1个酪氨酸磷酸化位点。整个蛋白质多肽链含有7个跨膜螺旋区,细胞外区和内区均为4个。亲水性/疏水性分析表明,黑熊Tas2R2蛋白质为疏水性蛋白,其亲水性区段所占比例较小。种间同源性比较显示,黑熊Tas2R2基因与大熊猫、猫、犬、马和小鼠的cDNA序列同源性分别为98.3%、91.5%、62.9%、58.7%、52.6%;氨基酸序列同源性分别为96.7%、85.5%、49.3%、44.7%、36.1%。黑熊、大熊猫、犬、猫、马和小鼠的Tas2R2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Tas2R2基因适合于构建不同物种间的系统进化树。

In this study, an approximately 1 019 bp DNA sequence of bitter taste receptor Tas2R2 gene was obtained by PCR and cloning technique from Asian black bear（ Ursus thibetanus）, genomic DNA（GenBank accession number： JQ239035）. This sequence contains a complete single 915 bp non-intron open reading frame, and encodes a 304 amino acid polypeptide. The isoelectric point（pl） of the protein is 9.49 and the molecular weight is 34. 80 kDa. Topology prediction showed that the Tas2R2 protein of Asian black bear contains three N-glycosylation sites, four serine phosphorylation sites, three tryptophan phosphorylation sites and one tyrosine site. The whole protein polypeptide chain comprised seven transmembrane helix regions, four extraceUular regions, and four intracellular regions. The T2R2 is a hydrophobic protein with less hydrophilic components. Homology analysis of the Tas2R2. gene sequences by Clustal W indicated that the cDNA sequence homologies of T2R2 gene of Asian black bear with those of giant panda, cat, dog, horse and mouse were 98. 3%, 91.5%, 62.9%, 58.7%, 52. 6%, respectively. The homologies of the corresponding amino acid sequences were 96.7%, 85. 5%, 49.3%, 44. 7%, and 36.1%. The Neighbor-Joining tree of the Tas2R2 gene exons from these six species was consistent with their species tree, indicating that the Tas2R2 gene also fits constructed molecular phylogenetic trees of different species.