| Summary: | <i>Foxl2</i> is an evolutionarily conserved female sex gene, which is specifically expressed in the ovary and mainly involved in oogenesis and ovarian function maintenance. However, little is known about the mechanism that regulates <i>Foxl2</i> specific expression during the ovary development. In the present study, we constructed the gonadal yeast one-hybrid (Y1H) library of <i>Chlamys</i><i>farreri</i> with ovaries and testes at different developmental stages using the Gateway technology. The library capacity was more than 1.36 × 10<sup>7</sup> CFU, and the length of the inserted fragment was 0.75 Kb~2 Kb, which fully met the demand of yeast library screening. The highly transcriptional activity promoter sequence of <i>C. farreri Foxl2</i> (<i>Cf-Foxl2</i>) was determined at −1000~−616 bp by dual-luciferase reporter (DLR) assay and was used as bait to screen possible transcription factors from the Y1H library. Eleven candidate factors, including five unannotated factors, were selected based on Y1H as well as their expressional differences between ovaries and testes and were verified for the first time to be involved in the transcriptional regulation of <i>Cf-Foxl2</i> by RT-qPCR and DLR. Our findings provided valuable data for further studying the specific regulation mechanism of <i>Foxl2</i> in the ovary.
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