| An increasing researches show that estrogen receptor and vitellogenin genes are quitesensitive to the environmental pollutants,especially endocrine disruptors, therefore, these twoindexes are often used for biomarkers to detect pollutants and play important roles in pollutionrisk prediction and evaluation of environment endocrine disruptors. ER and VTG gene have beenobtained in many organisms, but there is little information about the these two genes in marinepolychaete Perinereis aibuhitensis, which is commonly used as indicator of environmentalpollutants.In this study the full length cDNAs of ER and VTG from Perinereis aibuhitensis wereabtained based on the partial sequences of ER and VTG from the transcription library of P.aibuhitensis through RACE technique. The structures of these two cDNAs were analyzed by aseries of bioinformatics software. The mRNA expressions of these two gene in the body wall of P.aibuhitensis under B(a)P exposure were also determined using real-time fluorescencequantitative PCR. The results were as follows:1. The full length cDNA of P. aibuhitensis ER was2793bp, it included a114bp5’-untranslatedregion, a1398bp3’-untranslated region and1281bp open reading frame encoding426aminoacids. This sequence had been uploaded to NCBI and attained GenBank number for KF212194.Sequence analysis revealed that the protein included a DNA binding domain (88-163), whichcontains two zinc finger structures of C4type nuclear receptorCLVCGDVASGYHYGVASCEAC(91-111) and CPANGICEITKRRRKACQAC (127-146). Theamino acid sequence also contained a ligand binding domain (239-419). Bioinformatic analysisshowed that the molecular weight and isoelectric point of this encoded protein were47643.5Daand5.45, respectively. This protein did not have signal peptide and transmembrane domain,which indicated it belonged to hydrophilic protein. Multiple sequences alignment analysisrevealed that the homology between the amino acid sequence of P. aibuhitensis ER and somemolluscs, fishes, mammals and amphibians ER is between55%-59%.2. The full length cDNA of P. aibuhitensis VTG was5325bp,of which5’-untranslated regionwas47bp,3’-untranslated region was199bp, open reading frame was5079bp and encodedaprotein composed of1692amino acids. This sequence had been uploaded to NCBI and attainedGenBank number for KF673856.The deduced amino acid sequence had a vitellogenin domain(31-718) and a VWFD domain (1401-1613).Bioinformatic analysis showed that the molecularweight and isoelectric point of this encoded protein were185270.5Da and8.66, respectively.This protein contained signal peptide, but had no transmembrane domain, which indicated itbelonged to the secretory protein. Homology analysis indicated that the amino acid sequence ofVTG from P. aibuhitensis had low homology (21%-27%) to the proteins of some molluscs andfishes.3. The mRNA expression characteristics of ER and VTG of P. aibuhitensis under B(a)P exposure were detected through real-time fluorescence quantitative PCR. the results showed with the timeprolonging, the transcript level of ER mRNA in different dose of B(a)P was upregulated. In0.5μg/L B(a)P group the ER mRNA expression was0.64,1.33and2.32times respectively to thecontrol group, in5μg/L B(a)P group the transcript level of ER mRNA was1.21,1.38and1.61times respectively to the control group, and in50μg/L B(a)P group the mRNA expression of ERwas0.66,1.07and1.85times respectively to the control group. the increasing trend in lowestdose of B(a)P exposure(0.5μg/L) was the most obvious, but there was no significant differenceamong each group. The mRNA expression of P. aibuhitensis VTG significantly increased underthe exposure of B(a)P. In all B(a)P groups, the mRNA transcript level of VTG was upregulatedfirstly then downregulated with the time prolonging. In0.5μg/L B(a)P groups the mRNAexpression of VTG at14day was upregulated again compared with other groups. |
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