肌质网钙泵SERCA基因在中华绒螯蟹钙离子调控中的功能及其对蜕壳的影响
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Q 786;S 917.4

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中央级基本科研业务费 (2020TD36);江苏省农业重大新品种创制 (PZCZ201749);江苏省种业振兴“揭榜挂帅”项目 (JBGS〔2021〕031);江苏省现代农业产业技术体系 (JFRS-01-01)


Function of sarco/endoplasmic reticulum Ca2+-ATPase in calcium regulation and its effect on molting of Chinese mitten crab (Eriocheir sinensis)
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    摘要:

    中华绒螯蟹的蜕壳周期中具有高度复杂的钙调节机制,本研究探索了水体中不同Ca2+浓度 (40、80、160和320 mg/L)对中华绒螯蟹的Ca2+信号转导的影响,并对其中的关键因子肌质网钙泵(SERCA)进行基因干扰以探索其功能。结果显示:①养殖水体高浓度Ca2+可致Y器官和鳃组织中Ca2+信号通路相关基因SERCAPMCARyRNCX相对表达量上升,眼柄中MIH表达量下降,血淋巴中Ca2+与蜕皮激素水平上升,说明高浓度的Ca2+可促进中华绒螯蟹蜕壳。②利用RNAi技术干扰SERCA基因表达,普通水体和加入320 mg/L Ca2+的水体中中华绒螯蟹Y器官中PMCANCX基因表达量上升、RyR基因表达量下降且细胞内Ca2+浓度上升,表明SERCA基因在中华绒螯蟹维持细胞内Ca2+浓度和细胞内钙稳态机制中发挥重要作用。③长期干扰SERCA基因的表达可延长中华绒螯蟹的蜕壳间隔,且降低增重率与存活率。然而,高钙环境下长期干扰SERCA基因,相对于普通水体,蜕壳间隔缩短,存活率降低。研究表明,SERCA基因可能是通过对Ca2+信号的干扰而影响中华绒螯蟹的蜕壳。本实验将为中华绒螯蟹蜕壳机制的进一步研究提供基础参考。

    Abstract:

    The Chinese mitten crab (Eriocheir sinensis) is a vital freshwater economic crab in China. Its growth is limited by exoskeleton. In the process of growth and development, the exoskeleton must be shed periodically in order to complete its phased growth. A highly complex calcium regulation mechanism is needed in the molting cycle of E. sinensis. Sarco/endoplasmic reticulum Ca2+ATPase SERCA (SERCA) transports Ca2+ from the cytosol to the sarco/endoplasmic reticulum (SR/ER), where Ca2+ is mainly stored in cells. In order to improve the molecular mechanism of molting of E. sinensis, a series of functional analyses of the SERCA gene, the key factor of Ca2+ signal transduction, was carried out in this study. The experimental group prepared four kinds of culture water with different Ca2+ concentrations by adding analytical pure CaCl2 (40 mg/L, 160 mg/L, 320 mg/L) to the fresh water, and the control group without CaCl2. The relative expression of genes were tested using qRT-PCR method. The result showed that the relative expression of genes related to Ca2+ signal pathway SERCA, PMCA, RyR and NCX in Y-organs and gill tissues increased, the relative expression of MIH in eyestalks decreased. The concentration of Ca2+ and ecdysone in hemolymph increased tested using ELISA method. The results showed that high concentration of Ca2+ could promote the absorption of Ca2+ from the water environment and transport across cells to hemolymph through the joint regulation of SERCA, PMCA, RyR and NCX in the gills of E. sinensis, thus increasing the concentration of Ca2+ in the hemolymph. In Y-organs, the relative expression of genes related to the Ca2+ signal pathway was increased, and the Ca2+ signal was up-regulated, which was involved in the regulation of intracellular calcium homeostasis. Meanwhile, the MIH gene was down-regulated in the eyestalks. This series of reactions promoted the transformation of E. sinensis to the premolt stage, accompanied by an increase in the concentration of ecdysone in hemolymph. After injecting the ds RNA of SERCA gene to E. sinensis for 24 h, 48 h, 72 h and 96 h, the knock down efficiency of SERCA gene were 65.67 %, 55.5 %, 32.10 % and 15.43 %, respectively, which showed it could effectively knock down the expression of SERCA gene, meaning it can be used in the next experiment. After knocked down of SERCA gene by RNAi and no Ca2+ was added to the culture water, and knocked down SERCA gene in high calcium environment (320 mg/L of Ca2+ was added to the culture water), the relative expression of PMCA and NCX genes in Y-organs increased, while the relative expression of RyR gene decreased. The concentration of intracellular Ca2+ was tested using Fluo-3 AM probe and it was increased. It suggested that the SERCA gene played an important role in maintaining intracellular Ca2+ concentration and intracellular calcium homeostasis in E. sinensis. Continuous knock down of SERCA gene expression could significantly prolong (P < 0.05) the molting interval and decrease the weight gain rate and survival rate of E. sinensis. SERCA is the critial factor of Ca2+ signal transduction, which not only participates in the regulation of molting, but also plays an important role in many cellular functions, knock down of SERCA gene expression may cause intracellular calcium homeostasis imbalance, interfere with Ca2+ signal, and cause adverse effects on other physiological activities, thus reducing the survival rate of E. sinensis. Continuous knock down of the SERCA gene in a high calcium environment resulted in shorter molting interval but aggravated intracellular calcium disorder and decreased survival rate compared with continuous knock down of the SERCA gene group. The results of this experiment will provide an essential reference for the further study of the molting mechanism of E. sinensis. It suggests that SERCA gene plays an important role in molting of E. sinensis. The study will improve the molecular mechanism of molting of E. sinensis, provide basic reference for further study of the molting mechanism and genetic improvement of E. sinensis, and provide theoretical support in the exploration of scientific culture and improving culture efficiency.

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于俊杰,梁猛,陈雪,丁秀芳,周立水,冯文荣,唐永凯.肌质网钙泵SERCA基因在中华绒螯蟹钙离子调控中的功能及其对蜕壳的影响[J].水产学报,2024,48(10):109102

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  • 收稿日期:2022-04-11
  • 最后修改日期:2022-06-06
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  • 在线发布日期: 2024-09-30
  • 出版日期: 2024-10-01