Akt在中华绒螯蟹免疫中的功能

Akt作为PI3K-AKT 信号通路的核心分子，在细胞增殖、代谢、发育和存活等过程中发挥重要作用。本研究采用特异性引物及PCR技术，克隆获得了中华绒螯蟹Akt基因 (EsAkt)并进行生物信息学分析，实时定量PCR技术检测了EsAkt在中华绒螯蟹不同组织和不同免疫刺激后的表达水平，免疫荧光技术检测了EsAkt蛋白的细胞定位，双链RNA干扰技术分析了EsAkt干扰后凋亡基因的变化。结果显示，EsAkt分子包含PH结构域、中心催化结构域 (S_TKc)和羧基端疏水结构域 (S_TK_X) 3个保守的结构域。EsAkt在所有被检测的组织中呈现组成型表达，且在免疫相关组织 (如肝胰腺、鳃和血淋巴细胞)中的表达量显着高于胃和肠道。EsAkt蛋白主要以弥散状的形式分布在细胞质中。中华绒螯蟹注射脂多糖 (LPS)和嗜水气单胞菌后，显著诱导EsAkt转录本的表达，LPS免疫刺激12 h后，EsAkt响应达到峰值，为对照组的4.35倍，刺激48 h后，EsAkt的mRNA表达水平有所降低，但仍是对照组2.62倍。嗜水气单胞菌免疫刺激6 h后，EsAkt响应达到峰值，为对照组的9.05倍，刺激48 h后，EsAkt的mRNA表达量回落到正常水平。双链RNA干扰EsAkt后，EsAkt的mRNA表达量为对照组的0.38倍，而凋亡相关基因EsCaspase-3-like表达水平显著上调，为对照组的2.69倍。研究表明，EsAkt基因在中华绒螯蟹免疫中发挥重要作用，参与了机体对细菌引起的免疫应激过程。本研究丰富了中华绒螯蟹PI3K-AKT信号通路研究的基本资料，为进一步研究甲壳动物免疫防御机制奠定了基础。

Function of Akt in immunity in Chinese mitten crab (Eriocheir sinensis)

Eriocheir sinensis is one of the most important aquaculture species in China. With intensification of high-intensity feeding and degradation of the environment, the increasing diseases caused by bacteria infections have resulted in enormous economic loss in crab aquaculture. Akt is a core molecule of PI3K-AKT signaling pathway, which is highly conserved from invertebrate to vertebrate that plays essential roles in various physiological and pathological processes, such as cell proliferation, metabolism, development and survival. Although several Akts among invertebrates have been explored, the identities of Akt and their downstream effectors, especially in pathogen infection of crustaceans, remains poorly understood. In the present study, an Akt gene (designated as EsAkt) was identified in Chinese mitten crab E. sinensis, and the mRNA expression patterns of EsAkt in response to immune stimulations were investigated. The specific primers and PCR technology were used to clone and obtain the E. sinensis Akt gene (EsAkt). The expression level EsAkt in different tissues and after different immune stimulations were detected by real-time quantitative PCR (qRT-PCR). The cellular localization of EsAkt protein was detected by immunofluorescence assay. The changes of apoptotic gene after EsAkt RNA interference were analyzed by double stranded RNA interference. The results showed that EsAkt molecule contained the PH domain, the central catalytic domain (S_TKc) and the carboxy-terminal hydrophobic domain (S_TK_X). EsAkt was constitutively expressed in all tested tissues, and its expression in immunity related tissues (such as hepatopancreas, gills and blood lymphocytes) was significantly higher than that in stomach and intestine (P<0.01). Immunofluorescence assay results indicated that the oval hemocytes were observed in the bright field, and the cell nuclei were stained by DAPI in blue. The EsAkt-reactive areas were stained in green, and the positive green signals were mainly diffuse localization in the cytoplasm. After injecting lipopolysaccharide (LPS) and Aeromonas hydrophila into E. sinensis, the expression of EsAkt was significantly induced. After LPS immune stimulation for 12 h, the response of EsAkt reached the peak, which was 4.35 times that of the control group (P<0.01). After stimulation for 48 h, the mRNA expression level of EsAkt decreased, but it was still 2.62 times that of the control group (P<0.01); After 6 h of A. hydrophila immune stimulation, the response of EsAkt reached the peak, which was 9.05 times higher than that of the control group (P<0.01). After 48 h of stimulation, the mRNA expression level of EsAkt fell back to normal. Upon double stranded RNA interference with EsAkt, the mRNA expression level of EsAkt was 0.38 times higher than that of the control group (P<0.05), while the expression level of apoptosis related gene Escaspase-3-like was significantly up-regulated, which was 2.69 times higher than that of the control group (P<0.05). These results imply that EsAkt plays an important role in pathogen challenge by regulating the apoptosis related gene expression in E. sinensis, which enriches the basic data of the PI3K-AKT signaling pathway in E. sinensis and lays a foundation for further research on the immune defense mechanism of crustaceans.