School of Biology and Basic Medical Science, Soochow University, No.199 Ren-ai Road, Suzhou, 215123, Jiangsu, People's Republic of China.
Mol Biol Rep. 2012 May;39(5):5967-76. doi: 10.1007/s11033-011-1409-7. Epub 2011 Dec 30.
VASA is considered to be one of the most reliable molecular marker of germ cells. In order to study the Bombyx mori vasa-like gene (Bmvlg), the cDNAs of Bmvlg were cloned and sequenced, and the results showed that the Bmvlg gene from the fifth instar larval testes had four alternative splicing isoforms. The open reading frame (ORF) of the longest isoform was composed of 1,806 nucleotides encoding 601 amino acid residues and contained some known conserved domains. The other three isoforms had complete ORF, suggesting that the Bmvlg gene had several alternative splicing forms, completely different from that of Drosophila melanogaster. The results of sequencing demonstrated that the Bmvlg gene promoter had several elements conserved in eukaryotic and gonadal tissue-specific promoters. To detect the specificity of the Bmvlg promoter, a transient expression vector pSK-vlg-DsRed-polyA with a red fluorescent protein gene (DsRed), controlled by the Bmvlg promoter and a vector pIZT/V5-His-vlg-DsRed containing a Bmvlg fused with DsRed driven by the Bmvlg promoter, was constructed, respectively. Red fluorescence could be observed in some transfected BmN cells derived from silkworm ovaries and in the eggs injected with the vector pSK-vlg-DsRed-polyA, but red fluorescence could not be detected in the tissues of silkworm larva, after the transient expression vector was injected into blood, suggesting the Bmvlg promoter had gonadal tissue specificity. The transcription levels of Bmvlg in gonads of the fourth and fifth instar larvae were determined by fluorescent quantitative PCR, and the results revealed that the expression level of the Bmvlg gene in testes was slightly higher than that in ovaries. The expression levels of Bmvlg were lower in the fourth instar larva than that in the fifth instar larvae. Moreover, subcellular localization experiments showed that Bmvlg mainly existed in cytoplasm. These results provided new clues for understanding the function of the Bmvlg gene.
VASA 被认为是最可靠的生殖细胞分子标记之一。为了研究家蚕 vasa 样基因（Bmvlg），克隆并测序了 Bmvlg 的 cDNA，结果表明第五龄幼虫睾丸中的 Bmvlg 基因有四个选择性剪接异构体。最长异构体的开放阅读框（ORF）由 1806 个核苷酸组成，编码 601 个氨基酸残基，并包含一些已知的保守结构域。另外三个异构体具有完整的 ORF，表明 Bmvlg 基因有几种选择性剪接形式，与果蝇完全不同。测序结果表明，Bmvlg 基因启动子具有几种在真核生物和性腺组织特异性启动子中保守的元件。为了检测 Bmvlg 启动子的特异性，构建了一个带有红色荧光蛋白基因（DsRed）的瞬时表达载体 pSK-vlg-DsRed-polyA，该基因由 Bmvlg 启动子控制，以及一个含有 Bmvlg 融合 DsRed 的载体 pIZT/V5-His-vlg-DsRed，该基因由 Bmvlg 启动子驱动。可以在一些转染的来自家蚕卵巢的 BmN 细胞和注射了载体 pSK-vlg-DsRed-polyA 的卵中观察到红色荧光，但在注射了瞬时表达载体的家蚕幼虫组织中无法检测到红色荧光，表明 Bmvlg 启动子具有性腺组织特异性。通过荧光定量 PCR 测定了第四和第五龄幼虫性腺中 Bmvlg 的转录水平，结果表明 Bmvlg 基因在睾丸中的表达水平略高于卵巢。第四龄幼虫的 Bmvlg 表达水平低于第五龄幼虫。此外，亚细胞定位实验表明 Bmvlg 主要存在于细胞质中。这些结果为了解 Bmvlg 基因的功能提供了新的线索。
