罗氏沼虾蜕皮周期中内分泌调控和蜕皮信号通路相关基 因的表达分析.

Molting is the important physiological process in Macrobrachium rosenbergii. This work is aimed to explore the endocrine regulation of M. rosenbergii and the expression patterns of related genes in the molting pathway, and to reveal the molecular regulatory pathways of M. rosenbergii molting. The activities of molt-related enzymes (glutamine synthetase, β-N acetylglucosaminidase and chitinase）and ecdyhormone contents in the molting cycle of hepatopancreas and hemolymph in M. rosenbergii were determined, and the expressions of MrETHR, Mr-FTZ-F1, Mr-ECR, Mr-RXR, and Mr-MIH were detected by RT-qPCR. The results of enzymatic activity showed that glutamine synthetase was more active in hemolymphatic than in hepatopancreatic (P＜0.05). β-N acetylglucosaminidase had much higher pre-molting activity in hepatopancreatic and hemolymphatic than in the postmolt stage (P＜0.05). In the hepatopancreas, chitinase activity was the highest in the postmolt stage (P＜0.05). The content of ecdytropin in the hepatopancreatic and hemolymphatic was the lowest in the intermolt stage and the highest in the postmolt stage. The ORF length of Mr-ETHR gene was 1 173 bp, encoding 390 amino acids, and the sequence was relatively conserved. The ORF length of Mr-FTZ-F1 gene was 1 206 bp, encoding 401 amino acids. The Mr-ETHR, Mr-RXR and Mr-ECR expressed the highest in the postmolt stage, while Mr-FTZ-F1 was the highest in the premolt stage. The expressions of Mr-MIH reached the highest in the intermolt stage and the lowest in the postmolt stage. Cluster and correlation analyses showed that Mr-ETHR and Mr-RXR and Mr-ECR expression patterns were closely related and had a very significant positive correlation (r=0.7030, P＜0.01; r=0.8680, P＜0.01), Mr-FTZ-F1 and Mr-MIH expression patterns were similar and positively correlated (r=0.6665, P＜0.01), while Mr-FTZ-F1 was negatively correlated with Mr-ECR and Mr-ETHR (r=-0.8339, P＜0.01; r=-0.6275, P＜0.01). It was shown that the molting process of R. rosenbergii was regulated by glutamine synthetase, β-N-acetylaminoglucosidase and chitinase, and Mr-ETHR, Mr-RXR, Mr-ECR, Mr-FTZ-F1 and Mr-MIH were involved in regulating the molting process of M. rosenbergii, and Mr-ETHR, Mr-RXR and Mr-ECR were positively regulated in the molting signaling pathway, while MrFTZ-F1 and Mr-MIH were negatively regulated. Mr-ETHR, Mr-RXR and Mr-ECR play a positive regulatory role in the molt signaling pathway,while Mr-FTZ-F1 and Mr-MIH play a negative regulatory role. This study’s results provided a foundation for the study of molting regulation mechanism in the downstream of the molting signaling pathway in crustaceans. [ABSTRACT FROM AUTHOR]

蜕皮是罗氏沼虾重要的生理过程，为了探究罗氏沼虾蜕皮周期中内分泌调控与蜕皮通路中相关基因在蜕皮周期中 的表达模式，阐明罗氏沼虾蜕皮的分子调节通路。本研究测定了罗氏沼虾肝胰腺和血淋巴组织中蜕皮周期内蜕皮相关酶活性（谷 氨酰胺合成酶，β-N 乙酰氨基葡萄糖苷酶和几丁质酶）与蜕皮激素含量，并通过 RT-qPCR 分析了蜕皮信号通路中 Mr-ETHR、MrFTZ-F1 以及 RXR、ECR 和 MIH 基因在罗氏沼虾不同蜕皮周期内的表达模式。酶活测定结果表明，谷氨酰胺合成酶在血淋巴组织 中活力高于肝胰腺组织（P<0.05）；β-N 乙酰氨基葡萄糖苷酶在肝胰腺和血淋巴组织中蜕皮前期的活力远高于蜕皮后期（P<0.05）。 在肝胰腺中几丁质酶在蜕皮后期活力显著高于其他时期（P<0.05）。肝胰腺和血淋巴组织中蜕皮激素含量在蜕皮间期最低，蜕皮 后期达到最高，呈上升趋势。通过 PCR 扩增与测序验证获得了 Mr-ETHR、Mr-FTZ-F1 基因 ORF 全长序列，Mr-ETHR 基因 ORF 全 长 1 173 bp，编码 390 个氨基酸 ；Mr-FTZ-F1 基因 ORF 全长为 1 206 bp，编码 401 个氨基酸。荧光定量结果表明 Mr-ETHR、MrRXR 和 Mr-ECR 在蜕皮后期表达量达到最高，而 Mr-FTZ-F1 在蜕皮前期表达量达到最高。Mr-MIH 在蜕皮间期表达量达到最高，蜕 皮后期表达量最低，呈下降趋势。聚类分析及相关性分析结果表明 Mr-ETHR 与 Mr-RXR 和 Mr-ECR 表达模式相近且呈极显著正相 关（r=0.7030，P<0.01 ；r=0.8680，P<0.01), Mr-FTZ-F1 和 Mr-MIH 表达模式相近且呈极显著正相关（r=0.6665，P<0.01), 而 MrFTZ-F1 与 Mr-ECR 和 Mr-ETHR 呈极显著负相关（r=-0.8339，P<0.01 ；r=-0.6275，P<0.01）。研究表明罗氏沼虾的蜕皮过程受谷氨 酰胺合成酶、β-N 乙酰氨基葡萄糖苷酶和几丁质酶的调节，Mr-ETHR、Mr-RXR、Mr-ECR、Mr-FTZ-F1 和 Mr-MIH 参与调控罗氏沼 虾蜕皮过程，Mr-ETHR、Mr-RXR 和 Mr-ECR 在蜕皮信号通路中起正向调节作用，而 Mr-FTZ-F1 和 Mr-MIH 起负调节作用。本研究 结果为甲壳动物蜕皮信号通路下游基因的蜕皮调控机制研究提供了基础资料。 [ABSTRACT FROM AUTHOR]