The Function Of Bok In Zebrafish Glands Development

Zebrafish which has strong fertility, the embryo transparent and development speed, is an important model of molecular biology, developmental biology and genetics study. Members of the Bcl-2family are critical mediators of the delicate balance between survival and apoptosis in eucaryotic cells. Bcl-2family is a kind of very conservative protein, can be divided into three categories:anti-apoptotic Bcl-2family, pro-apoptosis Bax subfamily, and BH3-only the family. Bok (Bcl-2-related ovarian killer) is considered a pro-apoptotic member of the Bcl-2family, zebrafish contains bokl and bok2two subtype. In this study, we over-expression Bok in zebrafish, to understand the function of bok in the gonadal development, if we could get gonadal infertility of genetically modified fish,we can controlling genetically modified (GM) fish breeding and meet the standards of biological and environment safety for other GM species.The experimental materials is zebrafish, firstly,we use the RT-PCR technology and fluorescence quantitative PCR technology detection bokl and bok2expression in zebrafish different organs and embryonic development period of representation. The results show that bokl in zebrafish eyes, brain, intestine, ovarian, testis could detect the bokl mRNA high expressions; heart, kidney, and spleen also has weak expressed and no expression in liver; bok2expressions in zebrafish ovarian, testis, eyes, liver can be detected, and expression in the highest amount, and in other organs such as the brain, heart, spleen and kidney were not detected its expression. detection different early embryo developmental period, show that bokl in512cells begin to express, until90%epiboly it began to appear to high expression;after fertilization bok2has a high level of expression, after30%epiboly bok2expression reduction, until to90%epiboly the expression began to rise. Fluorescence quantitative PCR detection showed that in unfertilized embryo, bok2expression is much higher than bokl. Together, the bokl and bok2are maternal gene. bok mRNA expression is most abundant in the testis and ovaries, bok was, however, also readily detected in many additional organs. This indicates that bok may have a wider role rather than being limited to the godan developmet and form gamete.Then build beta-actin-bokl-GFP-nanos and beta-actin-bok2-GFP-nanos restructuring plasmid and with tol2mRNA co-injection zebrafish1-cell fertilized egg, fluorescence microscope observed that all the organs can detect green fluorescent, beta-actin promoter drive random integration. Get P0transgenic fish, mating with WT, feeding F1offspring to sexual maturity. Dissection the F1ovary, find that there is green fluorescence in the smaller cells, while not in the mature cells. Ovarian frozen sections observed that there was no morphology difference between the transgenic fish and wild fish. We need further study by staining, apoptosis analysis or using of specific gonad promoters to determine the function of the bok.