The Identification of a Cell Cycle Regulation Gene Cyclin E from Hong Kong Oysters (Crassostrea hongkongensis) and Its Protein Expression in Response to Salinity Stress

Abstract

Hong Kong oysters (Crassostrea hongkongensis) are an important marine bivalve with nutritional and commercial value. The expanded off-bottom farming scale in recent years makes the oysters more susceptible to exposure to abiotic stresses, such as salinity stress, an important environmental factor that has been proven to have significant effects on oyster growth and development. However, the molecular mechanism is still unclear. Cyclin E is an important protein in the process of cell cycle regulation that is indispensable for propelling G1/S phase transition in a dose-dependent manner. In order to investigate whether the salinity stress affects cyclin E expression in oysters, the cDNA sequence of C. hongkongensis cyclin E (Ch-CCNE) was isolated from a gill cDNA library, and the 2.8 kbp length cDNA fragment contained a complete open reading frame (ORF) encoding 440 amino acid residues. Ch-CCNE mRNA was highly expressed in the gonad and low in the adductor mussel, mantle, gill, labial palp, and digestive gland. The recombinant CCNE protein was expressed and purified in a pET32a(+)-CCNE/Escherichia coli BL21(DE3) system via IPTG induction and was used for generating mice anti-Ch-CCNE antiserums. Western blot analysis showed that the CCNE protein in the gill was maintained at low expression levels under either hypo- (5 ppt) or hyper- (35 ppt) salinity, and could be produced at high levels under appropriate salinity during a 10-day exposure period. The immuno-localization indicated that the Ch-CCNE protein was distributed in the nucleus. These results suggested that either hypo- or hyper-salinity stress could inhibit the CCNE expression of Hong Kong oysters and their negative impact on cell division and proliferation.