Cloning and expression of an actin gene in the haemocytes of pearl oyster(Pinctada fucata, Gould 1850)

摘要

An actin gene (designated pfactl) of pearl oyster, Pinctada fucata, was cloned from haemocytes by thetechniques of homological cloning and rapid amplification of cDNA ends (RACE). The full length of PfactlcDNA was 1608 bp in length, having a 5' untranslated region (UTR) of 82 bp, a 3' UTR of 395 bp, and an openreading frame (ORF) of 1131 bp encoding a polypeptide of 376 amino acids with a predicted molecular weightof 41.76 kDa and an estimated isoelectric point of 5.29. Sequence analysis revealed that Pfactl shared highsimilarity with other actins and was more closely related to vertebrate cytoplastic actins than muscle types.Phylogenetic analysis indicated that molluscan actins could also be generally grouped into two classes:muscle type and cytoplasmic type, although both are similar to vertebrate cytoplastic actins. Fluorescentreal-time quantitative RT-PCR was used to examine the expression level of Pfactl in haemocytes of P. fucataafter the challenge of Vibrio alginolyticus, and results showed that Pfactl exhibited stable expression in alltime points, indicating that Pfactl could be a suitable internal control for gene expression analysis inhaemocytes of P. fucata.