The Construction Of The So-Juy Mullet (Mugil Soiuy) ERα Gene Into The Yeast Two-Hybrid System To Explore One Possible Reason For The High Incidence Of Intersex In This Fish

So-juy Mullet (Mugil soiuy)is an important economical Fish in Coastal areas in china. A environmental survey around Bohai Bay found that the wild mullet in the incidence of intersex in different parts of a minimum of 10% up to 59 %, much higher than the other species investigated. Occurrence of intersex pike fishery resources of the region caused by the potential threat posed. previous study show that the occurrence of hermaphroditism in wild fish has been related to long term exposure to environmental estrogens(EES) polluted environment. Present knowledge show that The endocrine disruption fuction of most EES is mediated by the estrogen receptor(ER). Already, there is evidence that the ER binding affinity differ between species. The aim of this article is to establish a So-juy Mullet -specific ERαbinding screening method, and compareing the So-juy Mullet ER(sER) and ER binding activity of other species, from species differences in ER binding point of view, explain One possible reason for the phenomenon of the high incidence of intersex in So-juy Mullet.In this paper, 1113bp sERαestrogen receptor D, E/F fragments were amplicated from So-juy Mullet liver RNA useing RT-PCR and RACE methods, the sERαfragments were inserted into PGBT9 plasimd to build the plasmid PGBT9-sERαLBD, and then transfected this plasmid into Y190 yeast cell with pGAD424-TIF2 plasmid to construct the sERα-specific in vitro system in yeast. We tested the induceing activities of 9 chemicals with sERα, including three natural estrogens, estrone (E1), 17β-estradiol (E2)and estriol E3) , a synthetic estrogen, 17α-ethynylestradiol (EE2), three kinds of phenols, octylphenol(OP), nonylphenol(NP) and bisphenol A (BPA) and two phytoestrogens, 4′,5,7-genistein (Gensline)and zearalenone (Zearalenone).And then compared theβ-galactosidase inducing activity of E1,E2,E3, EE2 in sERα-TIF2 yeast, Medaka ERα-TIF2 (mERα-TIF2) yeast and rat ERα-TIF2 (rERα-TIF2) Yeast under the same condition.At last compared all the 9 chemicals′binding affinity between sERα-TIF2 yeast and mERα-TIF2 yeast. The results show that:1. The enzyme inducing activity of all 9 substances present a dose - response relationship, so the two-hybrid yeast can be used for the screening of EES which specific binding to the sERα. 2. E1,E2,E3, EE2 binding ability to rERαand mERα, EE2 and E3 combination with sERαand rERαall exsist significant significant difference, which further proof that there are some deviations to extrapolate a mammalian estrogen receptor binding activity in vitro data to other species, so it is necessary to build species-specific screening system.3. Dates show that the binding ability of E1, E2, E3, EE2 to sERαand mERαwere significantly different. The half maximal ERα-binding concentration (EC50) of E1 with sERαand mERαwere calculated at 3.69 and 16.98 nM respectively, with a relative estrogenic binding activity of 65.7% and 33.34% respectively, the binding affinity of E1 to sERαis 4.6 times higher than mERα. OP, BPA, Genistein, Zearalenone′s combination between the two ERαalso present significant differences, among which, binding of Genistein with sERαEC50 is 445.6 nM (mERα4134 nM), which is 9.28 times higher than mERαbinding ability. The data of four type Estrogenic substances indicates that So-iuy mullet is more sensitive than medaka from the view of the agonistic activity for estrogens, which would be one of possible reasons leading to its high incidence of intersex in Liaodong Bay.4. Has been widely detected in environmental samples, E1 has a relatively high RBA, and EC50 is 4.6 times lower than Madaka, we speculate that E1 may play an important role in the high incidence of intersex in wild So-iuy mullet, so that a further in vivo exposure experiment may designed in the future.These results show that the constructed sERαspecies specific yeast two-hybrid system can be used for the screening and evaluation of environmental estrogens which can bind to sERα. And compared to mERα,sERαis more sensitive to the tested substances, which may lead to high incidence of intersex in wild mullet.