Molecular Cloning And Characterrization Of FOXL2Gene And Its Response To Endocrine Disrupting Chemicals In Rare Minnow

Endocrine disrupting chemicals (EDCs) is due to human activities and released into the natural environment, which can disrupt hormone balance and have harmful effects on human or animal or even future generations, also can affect affect normal sexual differentiation in fish. Foxl2, a forkhead transcription factor, plays an important role iin ovarian differentiation in the early development of the female gonad in mammals and fish. How EDCs affect Foxl2expression is little known. As a very rare gene of ovarian differentiation in vertebrates Foxl2able to activate transcriptional and regulation of aromatase cypl9ala. Endocrine disruptors how to affect the expression of Foxl2still little known. In the present study, we isolated a Foxl2cDNA from the ovary of rare minnow Gobiocypris rams by RT-PCR and RACE technology successfully and cloning Foxl2gene sequence of full-length1698bp, open reading frame of921bp, encoding306amino acids,5’untranslated region of218bp,3’non-translated region of559bp. the molecular weight of34.2k Dalton, predicted isoelectric point9.17. Sequence alignment seen Foxl2forkhead domain and the C-terminal region with a high degree of similarity with other vertebrates protein sequence. Rare minnow and flounder, tilapia, medaka are more than83%in similarity, the highest sequence similarity of the zebra fish, up to96%. Compared with mammalian Foxl2sequence the rare minnow Foxl2lack poly alanine, glycine and proline repeat sequence. Establish the phylogenetic tree shows that the rare minnow Foxl2and fish as a sub-branch, and the evolutionary distance zebrafish.Real-time quantitative RT-PCR technology to examined Foxl2gene expression during early development stages and in different adult tissues. Among different adult tissues, Foxl2is mainly expressed in ovary, brain, gill, eye, and male spleen, almost undetectable in the testis. Take rare minnow juveniles and sampled once every four days, the gene expression levels of nine time points of Foxl2and cypl9ala were detected. Quantitative RT-PCR analysis showed that Foxl2expression was linear decrease and cypl9ala, the downstream target gene of Fox/2, had no correlation with Foxl2from18to50days post fertilization (dpf). Foxl2expression of the highest level after hatching30days, the expression level of the lowest after hatching50days. cypl9ala high expression at42days and18days after hatching. cypl9ala gene expression down to a lower level at22days and34days after hatching. Throughout the sampling period, cypl9ala gene expression pattern more stable than Foxl2. Analyzed Foxl2expression in G rarus juvenile following3-day exposure to17a-ethinylestradiol (EE2),4-n-nonyl-phenol (NP), and bisphenol A (BPA). Quantitative RT-PCR analysis showed that0.1nM EE2significantly up-regulated the expression of Foxl2gene (P<0.05),0.0lnM EE2led to a slight increase in Foxl2; However, lnM EE2almost no effect to Foxl2expression. While NP(10-1000nM) had no effect on Foxl2expression, which showed that the rare minnow Foxl2expression is not sensitive to NP. BPA exposure experiments, lnM BPA caused the Foxl2gene expression levels were significantly increased to1.94times the level of the control group (P<0.05),0.1nM and10nM BPA exposure concentration Foxl2transcriptional expression of no significant effect. The results showed that the different endocrine disrupting chemicals produce different effects on Foxl2gene expression. Altogether, these results provide basic data for further study on how Foxl2mediates EDCs impact on the sexual differentiation in G. rarus.