Induction Of Vitellogenin And Its Application In The Environmental Estrogen Detection

vitellogenin, a kind of macromolecule phospholipids glycoprotein, is synthetic under theregulation of estrogen,being the precursor of ovipary vertebrates yolk protein. In general,vitellogenin only exists in the mature female, providing the main nutrients for a developingembryo. But the males and immature female can also produce a large number ofvitellogenin,when was induced in estrogen or someting with estrogen effect. We cloned thecomplete sequnence of Paralichthys olivaceus Vg cDNA, and establish the method of Real-Time PCR for testing vitellogenin mRNA expression quantity of Paralichthys olivaceusliver.The young fish was induced by E2and plasticizer (DIOP). It can not only develop a wayto health aquiculture,but also develop a kind of basis to monitor the pollution in water. It canbring great application value and social benefit for water quality.In this study, the method of rt-pcr and RACE were used for Paralichthys olivaceusvitellogenin gene cloning.The full-length cDNA sequence, obtained by sequencing and splicing,with the length of5066bp sequence,has open reading frame of5037bp abd encode1677aminoacids and forecast its amino acid.It can be found by sequence a signal peptide sequence, asequence of vitellogenin area,an serine-rich region and three N-glycosylation sites exisitedindeduced sequnence. While multiple comparison analysis with other species, two conservativesequence of N-glycosylation sites and26conservative cysteine residues were deduced in thissequnence.It can be found by homology analysis and evolutionary tree analysis that theobtained sequnence belong to VgB.It was close to pleuronectiformes and perciformes with thehomology of80%above.It was relative close to cyprinodontiformes and gadiformes,withhomology between60%to70%.It was far away from amphibians, reptiles and birds.In this experiment, several different concentration (0.1μmol/L,1μmol/L,10μmol/L) ofE2and plasticizer (DIOP) were used to treat immature Paralichthys olivaceus for continuous35days. During the period of the experiment, the sample was taken at1h,3h,6h,12h1d,2d,3d,5d,7d,14d,21d,28d,35d. From real-time PCR results,it showed that the liver vitellogeninmRNA expression under the induction of E2and plasticizer,was both obvious. The level ofvitellogenin mRNA expression in the E2teatment groups were generally obvious higher thanDIOP treatment groups. In the three groups of different concentrations of induction of E2andDIOP, vitellogenin mRNA expression both showed a downward trend after rising first basicly,and a maximum was found on the third day.In addition,the immature Paralichthys olivaceus under the exposure of0.1μmol/L and1μmol/L E2and plasticizer were anatomied. And tissue sections from gill,liver and kidney weremade to observe the influence by estrogen. Experimental results showed that exposed to the twokinds of pollutants, the fish can produce tissue damage, and with the serious damage whenincreasing of concentration and the extension of time. Paraffin section showed that the gilllamella became bend and thin with intumescent terminal, and even became fractured withdeciduous and necrosis cell in the group of E2. Disordered arrangement of liver cell, swollenliver cell, fatty degeneration and cavitation appeared and renal tubular form becamedisintegrated, which renal tubule becoming thinner, lumen of renal tubule becoming bigger andglomerular structure being destroyed in the group of E2. While in the group of plasticizer,intumescent and bend terminal of the Gill lamella, abnormal cavity appeared with hyperplasiaand adhesion of the Gill lamella. In addition, slight fatty degeneration and cavitation alsoappeared, and renal tubular form was destroyed with damaged or invisible tubular cells.The results showed that Paralichthys olivaceus vitellogenin have good induction effectwhen is exposed by estrogen or estrogen material with effect,and can be detected by the methodof Real-Time PCR sensitively, accurately and efficiently.The result can make derectly responseof the degree of pollution. So vitellogenin of Paralichthys olivaceus can serve as a goodbiomarker for monitoring water pollution of estrogen.