|关键词||脊尾白虾 Eh基因 Iag基因 Sox9基因 蜕皮 性别决定|
甲壳动物的蜕皮是与其生长、变态和繁殖密切相关的重要生理过程。前期的研究表明蜕皮过程是由20-羟基蜕皮酮（20E）及其上游的激素、多肽和环境因子共同调控的，然而对于20E调控的下游基因以及其在蜕皮中的功能仍不清楚。蜕壳激素基因（eclosion hormone, EH），又称为羽化激素，最早是从飞蛾中分离得到的，随后在其他昆虫如烟草天蛾（Manduca sexta）、家蚕（Bombyx mori）、果蝇（Drosophila melanogaster）中都有报道。EH是由昆虫的腹神经细胞合成和分泌的一类神经肽，在昆虫蜕皮中起着重要的作用，至于EH在甲壳动物蜕皮中的作用了解得很少。本论文从脊尾白虾中获得了与昆虫的EH编码基因具有一定相似性的序列（命名为EcEH），其开放阅读框（ORF）为273 bp，可编码90个氨基酸；氨基酸序列包括一段疏水的信号肽，6个保守的半胱氨酸残基，具有保守的eclosion结构域。组织表达分析结果表明，EcEH 转录本主要在鳃、表皮及眼柄中表达。EcEH在脊尾白虾不同的蜕皮时期，其转录表达发生明显的变化，在蜕皮前期（late premolt）表达量最高，而在其它蜕皮时期，其表达量维持在较低水平，提示其可能与蜕皮相关。利用RNAi干扰技术和外源20-羟基蜕皮酮（20E）激素注射的方法进一步研究了EH在脊尾白虾蜕皮中的功能， 结果表明注射EcEH的双链RNA会导致蜕皮过程明显延迟，注射外源的20E可以明显加快蜕皮进程。以上研究结果说明EcEH与脊尾白虾的蜕皮密切相关。
胰岛素样促雄性腺激素（Insulin-like androgenic gland hormone, IAG）由甲壳动物雄性个体所独有的促雄性腺（Angrogenic gland, AG）分泌，在雄性甲壳动物的性别分化、雄性性征维持和精子发生中发挥着关键作用。IAG基因在沼虾、对虾、蟹类中均有报道， 但是对其功能的研究相对较少。本论文从脊尾白虾中克隆了IAG基因（EcIAG），并对其功能进行了分析。EcIAG基因的ORF全长531bp，可编码176个氨基酸；包括一段疏水的信号肽，6个保守的半胱氨酸残基，具有保守结构域（insulin-like IPRO16179）。EcIAG主要在雄性个体的促雄性腺（AG）及精巢（Te）中表达。原位杂交分析表明：EcIAG的转录本主要定位在促雄性腺细胞。对不同胚胎和幼体发育时期的转录表达分析结果表明，EcIAG在原肠期和仔虾P2期有着较高表达。为了解眼柄内分泌中心是否对EcIAG的表达具有调控作用，我们研究眼柄摘除对其表达的影响。结果表明：眼柄摘除后EcIAG的转录表达明显提高，说明眼柄中存在调控IAG表达的激素。原位杂交的相关研究为理解甲壳动物的性别分化调控通路提供了一定基础。
Sox9（SRY-box9）基因是一类与动物性别决定基因SRY具有高度序列相似性的转录因子。基于Sox9在哺乳动物等物种中参与性别调控过程，本研究尝试探讨EcSox9在促雄性腺中的表达分布情况及其与EcIAG之间的调控关系。本研究从脊尾白虾中克隆获得Sox9基因，其ORF全长672bp，编码243个氨基酸；推导的氨基酸序列具有保守的HMG BOX结构域。EcSox9 转录本在各组织中广泛存在，但在鳃及肝胰腺中表达量最高。EcSox9的表达随着胚胎和幼体发育时期的推移而呈现表达增加的趋势。EcSox9在摘除眼柄的脊尾白虾促雄性腺中表达量增加，表明眼柄中可能存在内分泌调控因子对EcSox9基因的表达具有调控作用。利用RNAi技术研究了EcSox9和EcIAG基因之间的关系，结果表明：注射EcSox9的双链RNA（dsSox9）时，EcIAG的表达量显著降低，表明EcSox9可能对EcIAG的表达存在正调控的关系；而在注射EcIAG的双链dsIAG时，EcSox9的表达量却显著升高，表明EcIAG的表达水平对EcSox9的表达存在反馈调控作用。相关研究结果为阐明Sox9在甲壳动物性别决定中的作用提供了重要资料。
Exopalaemon carinicauda is not only an important economic shrimp, but also a good experimental animal for biological studies of the crustaceans. Various physiological processes of crustacean, such as molting, growth etc, are regulated by interaction of multiple hormones. Molting is one basic physiological process of crustacean, which is closely related to growth and development. Knowledge on sex differentiation and sex determination is the basis of sex control in animals. In this paper, we identified several functional genes related to regulation of molting or sex development in Exopalaemon carinicauda and their functions were preliminarily analyzed. The main progresses are as follows:
Molting behavior is an important physiological process related to growth, metamorphosis and reproduction in crustacean. Previous studies have elaborated the molting process controlled by 20-hydroxyecdysone (20E) and upstream hormones, peptides and environmental factors which regulate 20E function. However, the downstream genes regulated by 20E and their functions during molting are still largely unknown. Eclosion hormone (EH) is a kind of neuropeptide that is regulated by 20E and triggers ecdysis behavior at the end of molting in insects. In the present study, an eclosion hormone like gene EcEH was identified from Exopalaemon carinicauda. The deduced amino acid sequence of EcEH contained a signal peptide, a typical eclosion domain and six conserved cysteine residues forming three putative disulfide bonds. EcEH was predominantly expressed in the epidermis, gills and eyestalk. In situ hybridization analysis showed that EcEH transcripts were localized in gills and eyestalks. During molting process, EcEH was mainly detected in the premolt stage. The expression level of EcEH in mid premolt stage could be up-regulated by exogenous 20E. Silencing of EcEH using double-stranded RNA delayed both the molting process and ecdysis behavior of E. carinicauda. Furthermore, injection of exogenous at mid premolt stage (D2) remarkably promoted the molting process and ecdysis rate of E. carinicauda. These data revealed that EcEH participated in the molting process in E. carinicauda and its expression might be regulated by 20E. To our knowledge, this was the first time to characterize the function of eclosion hormone in crustacean.
Insulin-like androgenic gland hormone (IAG), is secreted by the androgenic gland (AG), which is a specific organ of male crustaceans. AG plays a key role in the sex differentiation of male crustaceans, male sexuality and spermatogenesis. The IAG gene has been described in many crustaceans, including shrimp, prawns and crabs, but knowledge about its function is is still very limited. In this study, An IAG like gene (named as EcIAG) were identified in Exopalaemon carinicauda. The ORF sequence of IAG gene was 531 bp, encoding 176 amino acids. The amino acid sequence includes a hydrophobic signal peptide, 6 conserved cysteine residues, which form three disulfide bonds, and with two conserved domain (insulin-like IPRO16179). The tissue distribution of EcIAG was studied by RT-qPCR, which was specially expressed in AG and Testis. In situ hybridization showed that EcIAG was mainly localized in AG cells. The gene expression of IAG gene during different developmental stages was studied by RT-qPCR. The data showed that EcIAG was highly expressed in gastrul stage and postlarval stage P2. In order to know whethter the eyestalk has some hormones regulating the expression of EcIAG, we study the effects of eyestalk ablation on its expression. The results showed that the expression level of EcIAG was significantly up-regulated after eyestalk ablation, indicating that there were hormones in eyestalk regulating the expression of EcIAG. The related studies of in situ hybridization provide some information for understanding the regulation of sex differentiation in crustacean.
Sox9 (SRY-box9), a class of transcription factors with a high degree of sequence similarity to the sex determination gene SRY of the animals. In this study, we identified Sox9 gene in Exopalaemon carinicauda (named as EcSox9), and analyzed its function. The ORF sequence of Sox9 gene was 672 bp, encoding 243 amino acids. The deduced amino acid sequence includes conserved HMG BOX domain. Tissue distribution analysis showed that EcSox9 was widely distributed in the detected tissues, but it showed the highest expression level in gill and hepatopancreas. The expression of EcSox9 gene increased with the development of embryos and larvae. The expression of EcSox9 in the androgenic gland was apparently up-regulated after eyestalk ablation, which suggested that there were some endocrine factors negatively regulating the expression of EcSox9. RNAi technology was performed to test the possible regulatory relationship between the genes EcSox9 and EcIAG. Injection of double strand RNA of EcSox9 (dsSox9) reduced the expression of EcIAG in the androgenic gland, which indicated that EcSox9 might positively regulate the expression of ExIAG. Injection of double strand RNA of EcIAG (dsIAG) upregulated the expression of EcSox9, which indicated that the expression level of EcIAG had a feedback regulation on the expression of EcSox9.
|周丽红. 脊尾白虾蜕皮和性别调控相关基因的鉴定及功能分析[D]. 北京. 中国科学院大学,2016.|