关键词: T2R2基因, 克隆, 苦味受体, 猪獾, 分子进化分析

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 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 W indicated 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, respectively, 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.