关键词: 大熊猫, 苦味受体基因, 克隆, 序列分析

Abstract: Based on bitter taste receptor T2R2 gene sequence of domesticated dog (AB249685),one pair of primers were designed and used to amplify an approximately 1.1 kb DNA fragment from genomic DNA sample of giant panda by using PCR. The PCR products were ligated into the pMD-18T vector,and then transformed into competent cells of E.coli DH5α. The identified positive clone was sequenced. The result showed that the T2R2 gene of giant panda was 1 008 bp in length, and contained complete exon,and 915 bp,encoding 304 amino acid residues. The pI of the protein is 9.56 and its molecular weight is 34.90 kDa. The prediction of topological structure for the protein indicated that it contained 11 potential functional sites (three N-glycosylation sites,three potential protein kinase C phosphorylation sites,two N-myristoylation sites, one Casein kinase II phosphorylation site,one cAMP-and cGMP-dependent protein kinase phosphorylation site and one Nuclear Localization Signal site) with five sites observed only in giant panda,and the protein comprised seven transmembrane helix regions,and four extracellular regions and four intracellular regions. The T2R2 was a hydrophobic protein with less hydrophilic components which mostly were located on the intracellular regions. Alignment analysis revealed that the homology of T2R2 gene nucleotide sequence of giant panda with that of dog,cat,cattle,horse,chimpanzee and mouse is 92.65%, 91.12% , 85.64% , 86.73% , 85.20% , 72.59% , respectively, and the homologies of amino acid sequence is 86.73%, 85.20%, 74.67% , 78.62% , 75.66% , 60.53%, respectively. On the whole,the giant panda T2R2 gene was high evolutionarily conserved,but its protein presented more abundant functional sites than did those of other species. However,
the correlation between the characteristics of T2R2 gene and giant panda’s special diet needs to be further studied. The obtained sequences were submitted to GenBank,with accession NO. FJ812726.

Key words: Bitter taste receptor gene, Cloning, Giant panda (Ailuropodqa melanoleuca), Sequence analysis