Study On The Cytochrome P450 Enzymes And CYP Genes In Thais Clavigera

A large number of studies have demonstrated that imposex in marine gastropods is induced mostly by organotin pollutants. But the mechanism of imposex has not been identified. It has been accepted that the inhibition of cytochrome P450 aromatase by organotin compounds will result in the imposex phenomena. In this thesis, Marine gastropod, Thais clavigera, was chosen as the experimental material, because they are widespread, sensitive to organotin and have high imposex frequency. Considering P450 enzymes catalyze many reactions that are important in organism, cytochrome P450 enzymes and its genes in T. clavigera were studied. The results may contribute to further explain the imposex phenomena.Firstly, microsomes were respectively prepared from the visceral masses of male, female and imposex samples of T. clavigeras. The main components including cytochrome P450, cytochrome b₅ and NADPH-cytochrome P450 reductase in microsome cytochrome P450 enzymes were determined by spectrophotometry. The results indicated that the content of cytochrome b₅ in male and female was twice as that in imposex T. clavigeras, and the differences among them were significantly. Compared among the male, normal female and imposexed female snails, the content of cytochrome P450 and the activity of NADPH-cytochrome P450 reductase in imposex snails were more close to that in males. The result hinted that imposex snails were similar to males not only in morphology but also in some biochemical properties.Secondly, CYP genes were studied. CYP19 conserved regions were derived by aligning amino acid sequences by Clustal X. Degenerate primers were designed with Primer Premier 5. Genomic DNA and total RNA were isolated from T. clavigeras. The corresponding sequences were amplified by degenerate PCR and degenerate RT-PCR. Then the amplified fragments were ligated into pUCm-T vector and cloned. Their nucleotide sequences were determined. The result showed that a same fragment of 746bp was amplified from genomic DNA and total RNA. The BLAST programs were used for searching protein and DNA databases for sequence similarities. The BLAST result showed that it was a new sequence and similar to zebra fish DNA sequence and some fish reverse transcriptase. But it has small homology of CYP 19 gene sequences that have been in Genbank.In addition, sequences were amplified by RT-PCR with using degenerate primers that weredesigned against CYP4 sequences. However, the result of sequence analysis revealed that there was a 468bp fragment that existed in pleopod and viscera of T. clavigeras. It is a new sequence and its function is unknown. But it shows some sequence homology with the corresponding regions of insect CYP4. Its potential function needs further studies, such as RACE, blotting, gene expression to demonstrate.It is well-known that cytochrome P450 enzymes can be found in many types of tissues and organisms. They are very important to organisms. They play an essential role in drug metabolism, catalyze many physiological reactions such as carbon source assimilation, biosynthesis of hormones, and so on. In this paper, the experimental results of P450 enzymes were helpful to further understand the mechanism of imposex that caused by organotin compounds in T. clavigeras. And what we have studied on CYP genes could make foundation for further study on mollusks on the level of molecular biology.