Full-length Cloning And Related Function Study Of Cytochrome P450in Hyriopsis Schlegelii

In this study,(designated as Hs-CYP450) was cloned from by the method ofRACE PCR. Then, relevant biological softwares and online softwares were used tomake a preliminary analysis for Hs-CYP450genes sequence and primary, secondary,tertiary structures of protein. These results indicated that the Hs-CYP450cDNA afull length of1809bp consisted of an open reading frame (ORF) of1431bp encodinga protein of476amino acids; The content of leucine (Leu) accounted for9.5%in theprotein, but tryptophan is only0.8%; The hydrophobicity analysis showed that thisprotein was a hydrophilic soluble protein; The prediction of secondary structuresuggested that α helix (H) content is the most, accounting for46.22%and distributedevenly in the protein; β fold (E) is relatively less, about9.87%,located at both ends.The result of phylogenetic tree showed that Hs-CYP450gene sequence had a highhomology with other animals CYP gene, and the Crassostrea gigas shared the highestamong these species.The expression of CYP450mRNA in Hyriopsis schlegelii were measured byfluorescent real-time quantitative PCR. The results showed that the mRNA expressionof CYP450had significant differences in the different organization. The expressionlevel of CYP450in liver was the highest,and second higher in kidney, which wasrespectively5.3times and4.5times higher than that of foot. Followed by the gonad,heart,and in the blood,mantle, gills and adductor muscle were lower, and in the footwas the lowest.After stimulated by copper sulfate, potassium permanganate, trichlorfon,chlorine dioxide powder,we extracted the RNA and measured by fluorescent real-timequantitative PCR after reverse transcriptase at0h、24h、48h、96h. The results showedthat the expression of CYP450mRNA in Hyriopsis schlegelii were inhibited,andlower than control group at each times after stimulated by copper sulfate.Theexpression level of CYP450in Hyriopsis schlegelii at24h was the highest inliver,but,in kidney,this time is48h after stimulated by potassium permanganate.Onthe other way, when the concentration at20mg/L the level was the highest in the liverand kidney. After stimulated by trichlorfon,the gene also at24h had a highest expression in liver,and at48h the expression level was the highest in kidney.when the concentration at20mg/L the level was the highest in the liver and at10mg/Lin the kidney. The expression level of CYP450was higher than control group at24hafter stimulated by chlorine dioxide powder,but its not too obvious. And, when theconcentration arrived200mg/L,the level was the highest.