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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。
In order to study the phenomenon of precocious prawn sex, a Gtsf1 homologous EST sequence was found in the freshwater shrimp ovary expression profile constructed in laboratory. The cDNA sequence of Gtsf1 gene was cloned by RACE technology for 349 bp (GenBank accession number: KR349325) The open reading frame (ORF) was 240 bp, encoding 79 amino acids. Bioinformatics analysis predicted that the green shrimp Gtsf1 protein contains a zf-U11-48K domain, and found that the signal peptide-free, belonging to a non-secreted protein. Phylogenetic tree analysis showed that the Gtsf1 protein from shrimp was the closest to Lygus hesperus. Tissue expression analysis showed that Gtsf1 gene was highly expressed in the gonads, especially in the ovary (6 times that of the testis), while it was extremely low in other tissues. The expression of Gtsf1 gene at different developmental stages in ovary showed that Gtsf1 gene expression was low at the early stage of development. With the development of ovary, the expression level of Gtsf1 showed a significant upward trend (P <0.05), reached the peak at maturity and decreased significantly during the regression And recovered to a lower level (P <0.05). The results of embryo and post-embryo development show that the expression level of Gtsf1 gene is higher in embryogenesis stage and significantly increased and peaked at blastula stage (P <0.05), significantly decreased from gastrula stage to 10 days after metamorphosis Gene expression levels remained low (P <0.05). The above results show that Gtsf1 gene closely related to reproductive, especially in ovarian development, and a positive correlation trend. At the same time, Gstf1 gene may be mainly involved in the regulation of early embryonic development of the freshwater shrimp. This study was the first to clone Gtsf1 gene in crustacean and to analyze the spatial and temporal expression patterns of Gtsf1 gene in freshwater shrimp. The results will lay a foundation for further study on the function of Gtsf1 gene and regulation of gonadal development in young shrimp and other crustaceans Reproductive related research provide a reference.