Cheng Han-liang

SEQUENCE ANALYSIS OF THE RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACERS AND 5.8S RIBOSOMAL RNA GENE IN REPRESENTATIVES OF THE CLAM FAMILY VENERIDAE (MOLLUSCA: BIVALVIA)

Abstract The first and second internal transcribed spacer (ITS1 and ITS2) regions of the ribosomal DNA from six species, Meretrix meretrix, Cyclina sinensis, Mercenaria mercenaria, Protothaca jedoensis, Dosinia corrugata and Ruditapes philippinarum, in the family Veneridae were PCR amplified and sequenced. The size of the ITS1 sequence ranged from 522–900 bp, which was the largest range so far reported for a bivalve species, with GC contents from 57.66% to 65.62%. The size of the ITS2 sequence ranged from 281–412 bp, with GC contents from 65.21% to 67.87%. Extensive sequence variation and obvious length polymorphisms were noted for both regions in these species, and ITS2 sequence similarity was higher than that of ITS1 across species. The complete sequences of 5.8S ribosomal RNA gene were obtained by assembling ITS1 and ITS2 sequences, and the sequence length in all species was 157 bp. The phylogenetic tree of Veneridae clams was reconstructed by using ITS2 containing partial sequences of both 5.8S and 28S rDNA, and corresponding sequence information in Arctica islandica (Dahlgren et al. 2000) as an outgroup species. Tree topologies indicated that P. jedoensis has a closer relationship with M. mercenaria than with other species.

cDNA sequence and tissue expression analysis of glucokinase from liver of grass carp (Ctenopharyngodon idella )

A full-length cDNA coding glucokinase (GK) was cloned from the liver of grass carp ( Ctenopharyngodon idella ) by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods. The cDNA obtained was 2066 bp exclusive of poly (A) residues with a 1431 bp open reading frame encoding 476 amino acids. The GK protein has a calculated molecular weight of 53.7 kDa and isoelectric point of 5.11. Some conserved functional sites were found including one conserved hexokinase signature sequence Leu 156 -Phe 181 ; two N-linked glycosylation sites Asn 176 and Asn 214 ; one cell attachment sequence Arg 202 -Asp 204 ; one glycosaminoglycan attachment site Ser 455 -Gly 458 . The amino acid sequence has a high similarity to GK of other species, the percent identity compared with topmouth culter, common carp, human and rat are 98.1, 96.8, 80.3 and 79.8%, respectively. Tissue distribution of GK mRNA in brain, mesenteric adipose tissue, spleen, white muscle and liver of grass carp was analyzed by SYBR real-time fluorescence quantitative RT-PCR method using β-actin as an internal control for cDNA normalization. The result shows that the expression level of GK mRNA in liver was significantly higher than in mesenteric adipose tissue, spleen and brain (p<0.05). Relative expression profile of GK mRNA in liver normalized with β-actin level was 31, 454 and 649-fold compared with the levels in mesenteric adipose tissue, spleen and brain, respectively. Meanwhile, GK mRNA was not detected in white muscle. Key words : Ctenopharyngodon idella , glucokinase, full-length cDNA, tissue distribution, real-time PCR.