青虾精(卵)母细胞特有因子Gtsf1基因的克隆及其时空表达分析

MOLECULAR CHARACTERIZATION AND EXPRESSION ANALYSIS OF GTSFl IN THE ORIENTAL RIVER PRAWN MACROBRACHIUM NIPPONENSE

为了研究青虾性早熟现象,在实验室构建的青虾精卵巢表达谱中发现一个Gtsf1同源EST序列,采用RACE技术克隆获得青虾Gtsf1基因cDNA序列长349 bp(GenBank登录号:KR349325),开放阅读框(Open reading frame,ORF)为240 bp,编码79个氨基酸。生物信息学分析预测青虾Gtsf1蛋白包含1个zf-U11-48K结构域,并发现该蛋白无信号肽,属于非分泌蛋白。系统进化树分析表明青虾Gtsf1蛋白与昆虫纲的蝽类(Lygus hesperus)亲缘关系最近。组织表达分析结果显示,Gtsf1基因在性腺中表达很高,特别是在卵巢中表达最高(为精巢的6倍),其他组织中表达极低。卵巢不同发育时期表达结果发现,发育初期Gtsf1基因表达水平较低,随着卵巢的发育,该基因的表达水平呈现显著上升趋势(P<0.05),在成熟期达到最高峰,在消退期显著下降并恢复到较低水平(P<0.05)。胚胎和胚后不同发育时期结果显示,Gtsf1基因在胚胎发育早期表达水平较高,囊胚期时显著升高并达到峰值(P<0.05),从原肠期显著下降直至变态后第10天该基因表达水平维持较低水平(P<0.05)。以上研究结果表明Gtsf1基因与生殖密切相关,特别是在卵巢发育过程中有关,且呈正相关趋势。同时,Gstf1基因可能主要参与青虾胚胎早期发育的调控。研究首次在甲壳动物中克隆获得Gtsf1基因,并分析Gtsf1基因在青虾中的时空表达模式,研究结果将为进一步研究青虾Gtsf1基因功能和青虾性腺发育调控规律奠定基础,也为其他甲壳动物的生殖相关研究提供参考。

Gametocyte-specific factor 1 （Gtsf1） is involved in gonadal development and plays an important role in maintaining normal proliferation of germ cells in Mus musculus and Drosophila melanogaste. In order to study sexual precocity in Macrobrachium nipponense, the homologous cDNA sequence of Gtsf1 was screened from the ovary and testis differentially gene expression profile, and the full-length cDNA sequence was cloned by RACE technique （KR349325）. The length of its cDNA sequence was 349 bp, containing a 240 bp open reading frame （ORF）, encoded a 79-amino acid protein. MnGtsf1 protein contained a zf-U11-48K domain that shared highly with its homologues in other vertebrates. The sequence did not contain signal peptide, indicating it is a non-secreted protein. According to the phylogenetic tree analysis, MnGtsf1 has the closest evolutionary relationship with its homologous in Lygus hesperus. MnGtsf1 was exclusively expressed in the gonads, especially in ovary, which is 6 times higher than that in testis. The MnGtsf1 expression showed an increased trend with the ovarian development. MnGtsf1 expression in the early embryonic development was higher than that in the late embryonic development. The MnGtsf1 expression showed the highest in blastula stage, and its expression stayed at a low level from gastrula stage to the 10th day after metamorphosis. These results indicate that MnGtsf1 might be involved in gonadal development and early embryonic development.