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引用本文:宋柳,吕建建,王磊,孙东方,刘萍.三疣梭子蟹几丁质酶基因(PtCht6)的克隆及其在免疫中的功能分析.海洋与湖沼,2019,50(5):1080-1090.
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三疣梭子蟹几丁质酶基因(PtCht6)的克隆及其在免疫中的功能分析
宋柳1,2, 吕建建3,2, 王磊1,2, 孙东方2, 刘萍3,2
1.上海海洋大学 水产科学国家级实验教学示范中心 上海 201306;2.中国水产科学研究院黄海水产研究所 农业农村部海洋渔业可持续发展重点实验室 青岛 266071;3.青岛海洋科学与技术国家实验室 海洋渔业科学与食物产出过程功能实验室 青岛 266235
摘要:
几丁质酶(chitinase)在甲壳动物的蜕皮、消化和免疫等很多生物学过程中发挥重要功能,为深入探讨几丁质酶的免疫防御机制,本实验利用RACE技术克隆了三疣梭子蟹(Portunus trituberculatusPtCht6基因。该基因cDNA全长2736bp,开放阅读框(ORF)2103bp,编码700个氨基酸,具有几丁质酶GH18家族典型特征。利用实时荧光定量技术分析PtCht6在三疣梭子蟹不同组织、不同蜕皮阶段、不同病原感染以及低盐胁迫(11)下的表达特征,结果显示:PtCht6在各组织中均有表达且在肝胰腺中的表达量最高;在不同蜕皮时期的肝胰腺中,其表达量由蜕皮后期(A/B)、蜕皮间期(C)、蜕皮前期(D)依次递减(P<0.05);人工感染WSSV和副溶血弧菌后,PtCht6的表达量在肝胰腺中分别于12h、72h达到最大值,在血细胞中均于12h达到最大值,且相较于对照组,整体显著上调表达(P<0.05);低盐胁迫能够显著抑制该基因的表达,最高抑制73倍(P<0.05)。同时发现,低盐环境下感染WSSV后,该基因达到峰值的时间明显延迟(至少延迟12h,P<0.05)。该研究结果预示PtCht6作为免疫因子参与三疣梭子蟹的病原防御,且低盐胁迫在一定程度上抑制了该基因的免疫功能。
关键词:  三疣梭子蟹  几丁质酶  白斑综合征病毒(WSSV)  副溶血弧菌  低盐胁迫
DOI:10.11693/hyhz20190100001
分类号:Q78
基金项目:国家虾蟹产业技术体系,CARS-48号;泰山领军人才工程高效生态农业创新类计划,LJNY2015002号;国家自然科学基金面上项目,41576147号,41776160号;青岛市应用基础研究计划项目,17-1-1-95-jch号。
CLONING OF CHITINASE GENE (PtCht6) IN PORTUNUS TRITUBERCULATUS AND ITS FUNCTIONAL ANALYSIS IN IMMUNITY
SONG Liu1,2, Lü Jian-Jian3,2, WANG Lei1,2, SUN Dong-Fang2, LIU Ping3,2
1.National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China;2.Yellow Sea Fisheries Research Institute, Chinese Academy of Fisheries Science, Key laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Qingdao 266071, China;3.Qingdao National Laboratory of Marine Science and Technology, Functional Laboratory of Marine Fishery Science and Food Production Process, Qingdao 266235, China
Abstract:
Chitinase plays an important role in many biological processes in crustaceans, including molting, digestion, and immunity. To understand the immune defense mechanism of chitinase in Portunus trituberculatus, we successfully cloned a PtCht6 gene by RACE (rapid-amplification of cDNA ends). The property of the PtCht6 gene includes cDNA 2736bp, ORF 2103bp, encoding 700 amino acid residues of unstable protein, and features typical structural characteristics of GH18 chitinase family. We analyzed the expression characteristics of PtCht6 in different tissues, molting stages, pathogen infections, and low salt stress (11) of P. trituberculatus using real-time fluorescence quantitative technique. The results show that PtCht6 was expressed in all tissues of P. trituberculatus with the highest one in hepatopancreas. In the hepatopancreas of a different molting stage, the expression level decreased successively in the post-molt stage, inter-molting stage, and pre-molt stage (P<0.05). Under a normal circumstance, after artificial infection with WSSV (white spot syndrome virus) and Vibrio parahaemolyticus, the expression of PtCht6 in reached the maximum in hepatopancreas in 12h and 72h, and in blood cell in 12h and 12h, respectively. The expression was up-regulated except for a few time points (P<0.05). In addition, the low salt stress could inhibit significantly the expression of PtCht6 for up to 73 times (P<0.05), and the time when WSSV infection reached the peak was delayed significantly for at least 12h (P<0.05). Therefore, PtCht6 is an important immune factor of protecting P. trituberculatus from pathogen infection and a low salt stress could inhibit the immune function of the gene to some extents.
Key words:  Portunus trituberculatus  chitinase  white spot syndrome virus (WSSV)  Vibrio parahaemolyticus  low salinity stress
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