Expression of germ cell-less and Its Putative Regulatory Region in the Sea Cucumber, Apostichopus japonicus

Abstract

The germ cell-less gene belongs to the BTB/POZ protein-family, whose structural domain plays an important role in the occurrence of mammalian gonads as it specifically combines with other DNA proteins. In this study, a unigene annotated as germ cell-less (Ajgcl) was screened from the gonadal genome of Apostichopus japonicus (sea cucumber) through homology. The full-length Ajgcl cDNA sequence was obtained by 5' and 3' rapid amplification of cDNA ends (RACE). Subsequently, we analyzed the expression characteristic of germ cell-less in A. japonicus using several methods, including multiple sequence alignment, phylogenetic analysis, and real-time quantitative PCR (RT-qPCR). The results showed that germ cell-less attaches to the BTB/POZ superfamily. The full-length cDNA of Ajgcl was 2 316 bp, which contained a 725 bp 3'UTR, a 139 bp 5'UTR, and a 1 455 bp open reading frame (ORF), encoding a protein of 484 amino acids including a conservative BTB/POZ domain. RT-qPCR analysis revealed that germ cell-less was expressed in the ovary, testis, intestines, stomach, longitudinal muscle, respiratory tree, tube feet, and coelomocyte. Remarkably, the expression level in the ovary was the highest and 2.25 times that in the testis. Moreover, as a maternal factor, germ cell-less was expressed throughout embryonic development. During ovarian development, the expression of germ cell-less in growing stage 2 was slightly elevated, but when the ovum developed into mature stage 3, the expression level was significantly down-regulated. It was not until the end of gametogenesis that its expression level returned to its original level. The expression of germ cell-less did not change significantly throughout the developmental stages of the testis. In addition, the online tool Match was used to predict the transcription factor binding sites in the germ cell-less promoter of A. japonicus. Subsequently, a total of 12 transcription factor binding sites were projected, namely Oct-1, FOXD3, PAX-6, CRP, c-Myb and NF-1. Notably, there were four Oct-1 binding sites between -1890 and -608. The characteristics of germ cell-less gene promoter were obtained, and germ cell-less was shown to be a germ cell marker gene for A. japonicus. This study provides data for future studies related to germ cell development in A. japonicus.

Keywords

• apostichopus japonicus
• germ cell-less
• germ cell
• maternal factor