Study On The Molecular Mechanism Of Organotin-induced Imposex In Thais Clavigera

This study aimed to investigate the molecular mechanism leading to imposex in female gastropod mollusks by TBT contamination. Thais clavigera is a kind of subtidal benthic snail. Previous survey has shown that population show imposex, which is positively correlated with shipping density, so that the species was chosen as the experimental animal. In the study, some differences of protein and gene level between the male, normal and imposex female Thais clavigera from the harbor of Shantou were studied, main study aspects including total protein, SOD, CAT, LPO, DNA and RNA.Firstly, the normal and imposex females in Thais clavigera based on morphological character were investigated, the results showed 0.57% imposex females in the population in Nanao island where is polluted not seriously by organotin, whereas in the Guangao bay where is polluted severe, the imposex females in the population were almost up to 100%. It indicated imposex is closely related with the organotin pollution. The majority of imposex individuals were in their elementary and secondary stage, and there were few severe stage imposex individuals. It proved that the imposex phenomenon is a developing course.Secondly, we have studied the difference among the Thais clavigera individuals at protein level. It turned out that organotin pollution leads to some regular changes in Thais clavigera. The results by SDS-PAGE and isozyme analyses showed that protein expression in imposex individuals tended to approach that of male individuals along with some sexual organs like males. By detecting the activity or content of SOD, LPO and CAT, it showed the activity of SOD descended at the first stage of imposex and droped to the lowest point at the second stage, then went up subsequently; The change of the LPO content and the CAT activity was opposite to that of SOD, at the first and second stages went up, and then went down subsequently.Thirdly, male, normal and imposex females in Thais Clavigera from the harbor of Shantou were used for RAPD analysis with 40 random primers. It turned out 10 primers were able to amplify clear fragments and 170 bands were amplified, among them 40 were found to be polymorphic. The results proved the DNA of individuals from polluted region appears abnormalchange. It suggested that the different fragments have relations with the contamination of organotin. We also detected the individual's genomic DNA using the primer designed according to the fragment that come from differential display mKNA by PCR, it also proved that the organotin has caused the mutation of genomic DNA in Thais Clavigera and the effects were random.At last, a fragment of cDNA that display difference between female and male was founded by the differential display mRNA. Then a series of experiments such as fragment reclamation, secondary amplifying, cloning and sequencing were carried out. Based on the new fragment, we used RACE to get the whole gene, and a part sequence of the gene was obtained. The result of blast comparing showed a low homology with the present known nuclear acid sequence, it indicated that the differential display fragment maybe an unstudied gene.