Characterization of the foxl2 gene involved in the vtg expression in mud crab (Scylla paramamosain).

Abstract

Forkhead box protein L2 (Foxl2) is involved in multiple physiological processes, such as ovarian development, granulosa cell differentiation, ovarian follicle development, and oocyte growth. In this study, a Spfoxl2 gene encoded 530 amino acid protein with characteristic forkhead (FH) domain was identified from transcriptome data of mud crab Scylla paramamosain and validated the accuracy by PCR technology. Meanwhile, the orthologues of the Spfoxl2 gene in other 14 crustacean species were identified with the same method. Further multiple sequence alignment analysis revealed the Foxl2 was highly conserved, especially in the FH domain, even completely identical in several species. Besides, the semi-quantitative PCR (Sq-PCR) result showed Spfoxl2 gene was mainly expressed in the gonad (testis and ovary). Further quantitative real-time PCR (qRT-PCR) result demonstrated its expression level in the testis was significantly higher than that in the ovary (p<0.01). In addition, the qRT-PCR result showed that in zoea V, megalopa, and larval I, the expression level of Spfoxl2 in megalopa is the highest. In addition, a putative Foxl2 binding site was identified on the promoter region of Spvtg, and knockdown of Spfoxl2 mediated by RNAi technology increased the expression of Spvtg in the ovary, suggesting Spfoxl2 might be the upstream negative regulator of Spvtg. Overall, this study provided new insights into the role of Spfoxl2 in ovary development through regulating Spvtg expression in S. paramamosain.