MOLECULAR CLONING AND EVOLUTIONAL ANALYSIS OF NPY,UCP2,LPL AND HL GENE OF LARGEMOUTH BASS(MICROPTERUS SALMOIDES)

Largemouth bass(Micropterus salmoides) is a piscivorous teleost fish,found only in the fresh waters of North America.It is a good game fish in the United States,and a highly valued cultivated species in China.In order to study its lipid metabolism as well as its evolutional relationship with other percoids,we used RT-PCR method to get the partial cDNA sequences of NPY,UCP2,LPL and HL genes,and deduced their corresponding amino acid sequences.Rapid amplification of the cDNA ends(RACE) PCR was used to amplify the 3′-and 5′-end of NPY cDNA.The complete NPY cDNA sequence of largemouth bass was 664bp in length,composed of an open reading frame(ORF) of 300bp,encoding a protein of 99 amino acid residues.A search of the BLAST neuropeptide database revealed that the deduced neuropeptide sequence of largemouth bass NPY was very similar to the NPY of other species,such as European sea bass(Dicentrarchus labrax,93﹪ identity),orange-spotted grouper(Epinephelus coioides,91﹪ identity),house mouse(Mus musculus,86﹪ identity),and human(Homo sapiens,87﹪ identity).The obtained UCP2 core sequence was 737bp in length,which contains an open reading frame coding 245 amino acids.The length of the obtained LPL core sequence was 509bp,coding 169 amino acids.The length of the obtained HL core sequence was 666bp,coding 222 amino acids.We made the amino acid sequence comparisons among the four obtained genes of largemouth bass and other species,and got four phylogenetic trees by NJ method using software MEGA3.The phylogenetic tree built on the basis of conservative NPY gene,showed a right relationship of fish with mollusk,amphibia,aves and mammals,but it could not present accurate relationship of fish species.The phylogenetic tree built by less conservative UCP2 gene showed that largemouth bass and other percoids were farther away from mammals than the more primitive cyprinids.In order to make a further determination of the evolutional position of largemouth bass,we used the least conservative genes(LPL and HL) of the four to build phylogenetic trees,and found that largemouth bass was the closest to Chinese perch(Siniperca chuatsi),which was distributed only in the fresh waters of East Asia.These results support the close relationship of largemouth bass and Chinese perch.