Cloning Of P-glycoprotein Gene And Its Role In Dsp Toxin Resistance In Crassostrea Ariakensis

Objective:To explore the roles of P-glycoprotein (P-gp) in mechanism of diarrheic shellfish poisoning (DSP) toxins resistant in shellfish, P-gp gene in Perna viridis and Crassostrea ariakensis were amplified. The accumulation of okadaic acid (OA), a main component of DSP toxins, activity of P-gp and expression of P-gp gene in gills of C. ariakensis were detected after exposed to Prorocentrum lima, a dinoflagellate producing DSP toxins.Methods:P-gp gene in Perna viridis and C. ariakensis were amplified by RT-PCR. The homology analysis of the P-gp gene in related species was conducted using BLASTP program at NCBI. The levels of OA in the gill of C. ariakensis were determined with OKADAIC ACID (DSP) ELISA KIT (96T). P-gp activity was determined in isolated gill tissues of oysters in the presence or absence of P-gp inhibitors by rhodamine B efflux assay. Expressions of P-gp mRNA were analyzed by semi-quantitive RT-PCR using P-actin as a normalizing gene.Results:After exposed to P. lima, the level of OA in gills of C. ariakensis increased significantly with time. At 12,24,48 and 72 h after exposed to P. lima, the concentrations of OA in gills of C. ariakensis were 60.22,74.49,99.48 and 169.95 ng·g-1, respectively. OA exposure resulted in a 2.0～2.9-fold increase in P-gp activity as measured by the rate of efflux of rhodamine B and 1.36-1.74-fold increase in P-gp expression in gills of C. ariakensis.Conclusions:P-gp gene fragments were amplified successfully from P. viridis and C. ariakensis, sharing high homology with P-gp and related genes sequences in vertebrate and invertebrate. The increases in P-gp activity and P-gp mRNA expression suggested that P-gp had an important role in the resistant mechanism of C. ariakensis against DSP toxins.Keywords:Diarrheic shellfish poisoning; Crassostrea ariakensis; P-glycoprotein (P-gp)...