Molecular Mechanism Of Organotin-induced Imposex In Haliotis Diversicolor By Real-time Q PCR Array

Based on ESTs of TBT exposure and Vibrio Parahaemolyticus infection sequenced in previous study, and gene sequences from NCBI database for Haliotis diversicolor, 150 unigenes related to gonad development and stress response were selected by bioinformatics analysis, of which 140 sequences could be attributed to cellular components, molecular functions and biological processes with GO analysis. Total RNA were extracted from gill, gonad, hepatopancrea and pallium after injected with 1 μg/g(wt) TBT and TPT respectively in vivo at 6h, 24 h, 48 h of exposure. In order to identify suitable reference genes for m RNA expression analysis during organotin exposure, the expression stabilities of six reference genes were detected by q PCR. Meanwhile, 109 genes related to gonad development and stress induction were screened by primer design and with 64 genes performing real-time PCR verification, and 36 genes were sensitive to TBT and TPT. Finally, two reference genes and thirty organotin response genes were choosen to make a real-time Q PCR array. The results were as follows:Firstly, the expression stabilities of six reference genes(ACT-Actin, 18S-18 S r RNA, 28S-28 S r RNA, ELFA-elongation factor 1 alpha, RPL8-ribosomal protein L8 and TUA-tubulin alpha) were detected by q PCR and the results were evaluated by Ge Norm, Norm Finder and Ref Finder statistical algorithms. The results demonstrated that ELFA was validated as the most stable reference genes both in gill, gonad and all tested samples, while in hepatopancrea and pallium, it was 18 S and ACT respectively. In addition, all the six reference genes were used to calibrate the expression of RXR, a well-known gene involved in imposex caused by organotin. The results showed that inappropriate reference gene could highly influence the relative gene expression value and sometimes would cause total wrong interpretation of data.Secondly, m RNA expression of genes to TBT and TPT exposure in various tissues at different time course showed that, 36 genes showed significant changes to TBT and TPT exposure(p≤0.05). These 36 genes could be divided into three categories: immune related genes, detoxification enzyme coded genes and cell cycle related genes. Among them, there are ten immune related genes: macrophage expressed protein 1(MEP1), beta-thymosin(TMSB), Ras related nuclear protein(Ran), IL-1 receptor associated kinase 4(IRAK4), Ring-box protein(RBX), cathepsin L2(CTSL2), high mobility group protein 1(HMG1), prohormone convertase 1(PC1), cytidine deaminase(CDA), MIP-I gene for molluscan indoleamine 2,3-dioxygenase like protein I, exon 1-13(IDO1-exon); five detoxification enzyme coded genes: cytochrome c oxidase subunit III(COXIII), cytochrome c oxidase polypeptide Vb(COX5B), glutathione-S- transferase isoform(GST-iso), glutathione-S-transferase(GST), metallothionein(MT); and twenty-one cell cycle related genes: thioredoxin(TRX), zinc finger-DHHC-type containing Asp-His-His-Cys(ZDHHC), zinc finger protein 1(ZFP1), hemocyanin(Ab Hc), calreticulin(CAM), calmodulin 2(CAM2), calmodulin-HDr3CJ31 allele(Calp31), calponin-HDr2CJ39 allele(Calp39), stanniocalcin(STC), cell-cycle and apoptosis regulatory protein-1(CARP-1), transcription factor activating enhancer-binding protein 4(TFAP4), transforming growth factor beta-induced protein ig-h3 precursor(β-ig-h3), eukaryotic translation initiation factor 3 subunit 4(IF3-4), CCR4-NOT transcription complex m RNA(CNOT), ribosomal protein S20(RPS20), ribosomal protein L7(RPL7), 26 S proteasome non-ATPase regulatory subunit10(PSMD10), ubiquitin conjugating enzyme(UBI), CCAAT enhancer binding protein(CEBP), serine/argininerich splicing factor 4(SRSF4), retinoid X receptor(RXR). The significant changes of so many cell cycle-related genes containing five multi-functional genes(26 in total, accounting for 72.2%) suggested that organotin exposure remarkablely changed the cell differentiation, apoptosis and other important cellular processes in small abalone, which was likely to cause cell apoptosis and reprogramme in female gonad tissue, and hence to induce imposex.Eventually, twenty-one cell cycle related genes, five detoxification enzyme coded genes, four immune-related genes and two reference genes(EFLA and ACT) were finally selected to establish a 96 wells real-time microarray.