Screening and Identification of Transcription Factors Potentially Regulating Foxl2 Expression in Chlamys farreri Ovary

SIMPLE SUMMARY: Foxl2 generally presents a sexually dimorphic expression pattern in animal gonads and is highly expressed in the ovary. However, few studies on the transcriptional regulation of Foxl2 have been documented. To understand the transcriptional regulating of Foxl2 high expression in the ovary, we used the Y1H system, a high throughput approach, for the first time to screen the transcription factors binding to the high transcriptional activity region of Foxl2 promoter in Zhikong scallop (Chlamys farreri) gonads. In the present study, the highly transcriptional activity promoter sequence of Cf-Foxl2 was determined at −1000~−616 bp and 11 candidate factors were verified to involve in Cf-Foxl2 transcriptional regulation. Our findings provided valuable data for better understanding the specific transcriptional regulation mechanism of Foxl2 in the ovary and would further assist in the breeding of aquacultural bivalves. ABSTRACT: Foxl2 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 Foxl2 specific expression during the ovary development. In the present study, we constructed the gonadal yeast one-hybrid (Y1H) library of Chlamys farreri with ovaries and testes at different developmental stages using the Gateway technology. The library capacity was more than 1.36 × 10(7) 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 C. farreri Foxl2 (Cf-Foxl2) 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 Cf-Foxl2 by RT-qPCR and DLR. Our findings provided valuable data for further studying the specific regulation mechanism of Foxl2 in the ovary.