A Selenium-dependent Glutathione Peroxidase In The Japanese Scallop, Mizuhopecten Yessoensis: Its CDNA Cloning, Promoter Sequence Analysis And MRNA Expression

The Mizuhopecten yessoensis, is an economically important marine mollusc found in the cold seas along the coasts of the northern islands of Japan, the northern part of the Korean Peninsula, and the Sakhalin and Kuril islands. In 1982, M. yessoensis (332 individuals) was introduced into China from Japan for aquaculture production, and rapidly became a favored aquaculture species in Dalian due to its large size and high market value. However, problems with high mortality, poor growth and seed production have heavily impact on commercial M. yessoensis farming for more than 10 years, and have caused serious economic losses to the scallop aquaculture industry in China. An improved understanding of the immune system of scallops and brief introduction of MyGPx will facilitate the effective management of disease and the development of sustainable scallop cultures.The complete coding sequence for Mizuhopecten yessoensis GPx(MyGPx) (GenBank accession no: HQ433529) was obtained from the mantle edge by gene cloning method using RT-PCR and the RACE technology. The full-length 1081 nt MyGPx mRNA contained a 28 nt 5′untranslated region (UTR), a 603 nt open reading frame and a 450 nt 3′UTR containing a polyadenylation signal (AATAAA). Multiple sequence alignment revealed that the deduced amino acid sequence of MyGPx has significant identities with other invertebrate and vertebrate selenium-dependent glutathione peroxidases. Our phylogenetic analysis suggested that MyGPx, together with the GPx of two other bivalves, Pinctada fucata and Hyriopsis cumingii, is similar to the GPx1 and GPx2 enzyme isoforms.A 1,686 bp PCR fragment, obtained by genome walking method, contained a 58 bp MyGPx cDNA sequences and a 1,628 bp MyGPx 5′-flanking sequence. TRANSFAC and AliBata 2.1 software were used to search for potential TFBSs in the putative promoter region. Nine putative SNPs were detected by aligning the 1,467 bp-gene fragments obtained from 15 individual scallops. The putative SNPs would provide more potential molecular locus for the molecular breeding program of Japanese scallop.Quantitative Real time PCR (qRT-PCR) was employed to measure GPx mRNA expression in adult tissues and monitor mRNA expression patterns during embryonic development and following stimulation by the bacteria Vibrillo anguillarum.The results showed that mantle own the highest expression level of MyGPx in all the adult tissues monitored, the expression reached the highest level in the trochophore and reached a maximal level at 12h after bacteria(lVibrillo anguillarum)stimulation. Collectively, the results suggest that MyGPx fulfils an important function during M. yessoensis development and may be an important immune effector in adult molluscs.