Goldfish (Carassius Auratus) Vitellogenin CDNA Cloning And MRNA Expression And Its Application In Environmental Estrogen Screening

Vitellogenin (VTG) is an important biomarker for monitoring environmental estrogens (EEs) in aquatic environment. VTG mRNA in male fish is a new kind of biomarker. In our country, we often choose community and chemistry methods to detect EEs and reports of molecular biology methods in detecting EEs are seldom. Monocrotophos (MCP), a kind of organophosphorus pesticide in common use in the past, has made serious pollution of pesticide residues. We use goldfish treated by MCP as a model animal to study the influence to the expression of VTG mRNA.In our laboratory, Bing Xin has isolated and purified vitellogenin in serum of 17β-estradiol treated goldfish and measured molecular weight of vitellogenin of 440kDa by native-PAGE. In addition, MCP, with concentration of 0.01 mg L-1, 0.01 mg·L-1,0.10 mg·L-1,1.00mg·L-1 can induce the production of vitellogenin in male goldfish serum. Based on the production, we use RT-PCR to detect expression of goldfish VTG mRNA and use the segment of partial cDNA as the template to synthesize DIG labeled RNA probe.Goldfish is purchased from a local ornamental fish supplier. All fish were maintained indoor in dechlorinated tap water for two weeks. Total liver RNA was extracted from the liver by Trizol kit. According to the conserved domains of amino acids, we design degenerate primers for RT-PCR and get a segment of 1135bp. Special bands was purification and cloned in EcoliⅠ. Clone was sequenced and compared with other species in Blast on NCBI net. As a result, this sequence has homology of 94 percent with common carp (Cyprinus carpio), 93% percent with goldfish (Carassius auratus) and 90% percent with fathead minnows (Pimephales promelas). In the present study, part of goldfish vitellogenin genes have been isolated and used as a biomarker to evaluate the influence of MCP.The induction of vitellogenesis is a complex process requiring coordinated control and expression of many hepatic gene products, such as vitellogenin (VTG). The authors have isolated a partial cDNA encoding goldfish VTG by RT-PCR. This cDNA sequence showed high homology with the VTG of other fishes. DIG-labled RNA probe was synthesized and Northern blot analysis yielded a VTG transcript from MCP treated goldfish hepatic tissue. We develop a new model of molecular biology to screen environmental estrogens.