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

doi:10.13560/j.cnki.biotech.bull.1985.2023-0169

摘要/Abstract

摘要：

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、Mr-FTZ-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、Mr-RXR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Mr-FTZ-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。

关键词: 罗氏沼虾, 蜕皮激素, 蜕皮时期, 蜕皮信号通路

Abstract:

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 Mr-ETHR, 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 Mr-FTZ-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.

Key words: Macrobrachium rosenbergii, molting hormone, molting period, molting signaling pathways

丁丽, 都婷婷, 唐琼英, 高权新, 易少奎, 杨国梁. 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[J]. 生物技术通报, 2023, 39(9): 300-310.

DING Li, DU Ting-ting, TANG Qiong-ying, GAO Quan-xin, YI Shao-kui, YANG Guo-liang. Analyses of Endocrine Regulation and Expression of Genes Related to the Molting Signaling Pathway in the Molting Cycle of Macrobrachium rosenbergii[J]. Biotechnology Bulletin, 2023, 39(9): 300-310.

图/表 7

图1 节肢动物蜕皮调控过程示意图 MIH受体是G蛋白偶联受体，当MIH与受体的结合时，细胞内cAMP增加，激活了细胞膜上的Ca2+通道，Ca2+进入细胞内并激活了钙调蛋白（CaM），CaM去磷酸化一氧化氮合酶，激活NOS，以精氨酸为底物形成NO，而后激活鸟苷酸环化酶I型（GC-I），GC-I使GTP转化为cGTP，激活细胞内的cGTP依赖的蛋白激酶，活性的PKA进入细胞核，调节蜕皮激素20E的合成，而20E入核后，与异源二聚体（ECR/USP/RXR）结合后，启动早期应答基因，如E75、E74和Br-C等，随后调控早晚期基因，如HR3、HR4和HR38等，最后这些早晚期应答基因调控晚期应答基因，如FTZ-F1，最终完成蜕皮。内分泌Inka细胞分泌蜕皮启动激素（ETH）作用蜕皮启动激素受体（ETHR），启动蜕皮

Fig. 1 Schematic diagram of the regulation process of molting in arthropods MIH receptor is G protein-coupled receptor. When MIH binds to the receptor, intracellular cAMP increases, activating Ca2+ channels on the cell membrane, and Ca2+ enters the cell and activates calmodulin（CaM）. CaM dephosphorylates nitric oxide synthase activates NOS, then NO is formed with arginine as a substrate, and then this activates guanylate cyclase type I（GC-I）, GC-I converts GTP to cGTP, and activates cGTP-dependent protein kinases in cells. The active PKA enters the nucleus to regulate the synthesis of ecdyhormone, and after 20E enters the nucleus, it binds to heterodimer（ECR/USP/RXR）to initiate early response genes, such as E75, E74 and Br-C, and then regulates early-late genes, such as HR3, HR4 and HR38, and finally these early and late response genes regulate late response genes, such as FTZ-F1, and finally complete molting. Endocrine Inka cells secrete ecdy-initiating hormone（ETH）acting on the ecdy-initiating hormone receptor（ETHR）, initiating molt

表1 实验所用引物序列

Table 1 Primer sequences used in the experiment

引物名称Primer name	引物序列Primer sequence（5'-3'）	产物长度Product length/bp	退火温度Annealing temperature/℃
FTZ-F1-F-1	CCTATTATGCTGCGGGAGT	649	55
FTZ-F1-R-1	CACCGTTGATGTGCTTGTA	649	55
FTZ-F1-F-2	TGTCCGTACTGTCGCTTTC	627	52
FTZ-F1-R-2	ACCAACTCCCGCAGCATA	627	52
FTZ-F1-F-3	CCTATTATGCTGCGGGAGT	595	55
FTZ-F1-R-3	GAAGCTCTGGCAGTAAATCC	595	55
ETHR-F-1	GTCCCGTTGCTGGTTCTT	535	55
ETHR-R-1	ATGACGCTGACCCTGTTG	535	55
ETHR-F-2	CGATGACGCTGACCCTGT	537	55
ETHR-R-2	AAGCCAAGGCGATAACAG	537	55
ETHR-F-3	ATAGTGCAGCCGCAGATT	635	53.5
ETHR-R-3	GGATCACCTGCACCAACGTA	635	53.5
RTFTZ-F1-F	GGATCACCTGCACCAACGTA	120	57
RTFTZ-F1-R	GGAAACGATCTGCGAACTGC	120	57
RTETHR-F	GTCCCGTTGCTGGTTCTT	165	55
RTETHR-R	GTCTCGCTCGCATTTGTG	165	55
RTMIH-F	AGCCCTGAGTGTCTGTCC	123	57
RTMIH-R	CCTTGCGTTGTCTGGTT	123	57
RTRXR-F	GATCGGCAGTCCCCTTTGAA	109	57
RTRXR-R	TTGGACACACTGGGAGAAGC	109	57
RTECR-F	AGAGCCGCATAAAGTGGAGA	134	57
RTECR-R	CTCAGGTCGGTCAGGATGTT	134	57
18S-F	TATACGCTAGTGGAGCTGGAA	313	60
18S-R	GGGGAGGTAGTGACGAAAAAT	313	60

图2 罗氏沼虾蜕皮周期中蜕皮相关酶活性变化 C：蜕皮间期；D：蜕皮前期；E：蜕皮期；AB：蜕皮后期；不同小写字母代表显著差异（P<0.05），下同

Fig. 2 Dynamics in molting-related enzyme activity in M. rosenbergiiat at different molting cycles C: Intermolt stage. D: Premolt stage. E: Molt stage. AB: Postmolt stage. Different letters indicated significant differences（P<0.05）, The same below

图3 罗氏沼虾肝胰腺与血淋巴中不同蜕皮时期蜕皮激素含量变化

Fig. 3 Dynamics of ecdyhormone content in hepatopancreas and hemolymph in M. rosenbergii at different molting stages

图4 基于NJ法构建的Mr-ETHR（A）、Mr-FTZ-F1（B）基因系统发育树

Fig. 4 Phylogenetic trees of Mr-ETHR（A）and Mr-FTZ-F1（B）based on NJ method

图5 在不同蜕皮时期内Mr-ETHR、Mr-FTZ-F1基因在肝胰腺和血淋巴组织中的表达情况

Fig. 5 Expressions of Mr-ETHR and Mr-FTZ-F1 in hepatopancreatic and hemolymphatic tissues at different molting stages

图6 在不同蜕皮时期内罗氏沼虾ETHR、FTZ-F1、RXR、ECR和MIH基因的表达情况

Fig. 6 Expressions of Mr-ETHR, Mr-FTZ-F1, Mr-RXR, Mr-ECR and Mr-MIH genes in M. rosenbergii at different molting cycles

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