日本对虾Bcl-2基因的cDNA克隆与抗寒功能研究  

CLONING OF CDNA OF BCL-2 GENE OF MARSUPENAEUS JAPONICUS AND ITS ROLE IN COLD RESISTANCE

作　　者：邵慧鑫 任宪云[2,3] 于振兴 李健 刘萍[2,3] SHAO Hui-Xin;REN Xian-Yun;YU Zhen-Xing;LI Jian;LIU Ping(National Demonstration Center for Experimental Fisheries Science Education,Shanghai Ocean University,Shanghai 201306,China;Key Laboratory of Sustainable Development of Marine Fisheries,Ministry of Agriculture and Rural Affairs,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao 2660712,China;Laboratory for Marine Fisheries Science and Food Production Processes,Pilot National Laboratory for Marine Science and Technology,Qingdao 266237,China)

机构地区：[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试点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266237

出　　处：《水生生物学报》2022年第8期1160-1167,共8页Acta Hydrobiologica Sinica

基　　金：山东省农业良种工程重大课题“优质高抗对虾突破性新品种选育”(2019LZGC014);中国水产科学研究院院级基本科研业务费“海水池塘生态养殖创新团队项目”(2020TD46)资助。

摘　　要：为研究B淋巴细胞瘤-2(B-cell lymphoma,Bcl-2)基因在内源性细胞凋亡通路中发挥的重要调控作用,实验利用cDNA末端快速扩增(RACE)技术克隆获得日本对虾(Marsupenaeus japonicus)MjBcl-2基因cDNA全长序列,并对其序列进行生物信息学分析;利用实时定量PCR(qPCR)技术分析了MjBcl-2在不同组织、不同水温胁迫及RNA干扰后的表达水平;同时利用TUNEL技术,检测MjBcl-2干扰后的细胞凋亡情况。结果显示:日本对虾MjBcl-2 cDNA全长为2432 bp,开放阅读框长度为726 bp,共编码241个氨基酸,分子量为26.80 kD;结构域预测分析表明MjBcl-2含有Bcl-2家族典型的保守结构域;多序列比对以及进化树构建结果表明,MjBcl-2与其他物种的相似度较高,保守性较强。定量PCR结果显示,MjBcl-2在日本对虾各个组织中均有表达,肌肉中表达量最高,鳃次之,心脏中表达量最低。低温(10℃和16℃)胁迫下日本对虾鳃和肝胰腺Bcl-2基因的表达量逐渐上升,在72h到达最高点;在干扰Bcl-2基因后,各个温度Bcl-2基因的表达量均下调,细胞凋亡基因Caspase-3的表达量显著上调(P<0.05)。TUNEL检测结果表明,RNAi组和NC组(对照组)随着温度的降低,凋亡细胞数量不断增加,在28℃处理组中有少量凋亡细胞,RNAi组与NC组凋亡细胞的数量没有明显的变化,在10℃低温处理组中,RNAi组凋亡细胞的数量明显高于NC组;12h后,28℃NC组、28℃RNAi组、10℃NC组和10℃RNAi组中鳃组织的细胞凋亡率分别为1.73%、2.35%、21.59%和33.70%。研究表明,MjBcl-2在日本对虾应对低温胁迫中发挥重要作用。As an important apoptosis regulatory gene,Bcl-2 gene plays an important role in the endogenous apoptosis pathway.In this study,the full-length cDNA sequence of MjBcl-2 gene was cloned by rapid amplification of cDNA ends(RACE)and analyzed by bioinformatics;Real time quantitative PCR(qPCR)was used to analyze the expression level of MjBcl-2 in different tissues of Marsupenaeus japonicus after different temperature stresses and RNA interferences;TUNEL technique was used to detect the apoptosis after the interference of MjBcl-2.The results showed that the full length of MjBcl-2 cDNA was 2432 bp,the open reading frame was 726 bp,encoding 241 amino acids with a molecular weight of 26.80 kD;Domain prediction analysis showed that MjBcl-2 contained typical conserved domains of Bcl-2 family.The results of multiple sequence alignment and phylogenetic tree construction showed that MjBcl-2 was highly conserved and similar to other species.Quantitative PCR results showed that MjBcl-2 was expressed in all tissues of M.japonicus,with the highest expression in muscle,the second in gill and the lowest in heart.Under the stress of low temperature(10℃and 16℃),the expression of Bcl-2 gene in gill and hepatopancreas of M.japonicus increased gradually,and reached the highest point at 72h;After interference with Bcl-2 gene,the expression of Bcl-2 gene was down-regulated and Caspase-3 gene was up-regulated at all temperatures(P<0.05);There were only a few apoptotic cells in the 28℃treatment group.TUNEL results showed that the number of apoptotic cells increased with the decrease of temperature in RNAi group and NC group(control group).There were a small number of apoptotic cells in 28℃treatment group,but there was no significant change in the number of apoptotic cells in RNAi group and NC group.The number of apoptotic cells in 10℃hypothermia treatment group was significantly higher than that in NC group;After 12h,the apoptosis rates of gill tissue in 28℃NC group,28℃RNAi group,10℃NC group and 10℃RNAi group were 1.73%,2.35

关 键 词：BCL-2 低温胁迫 组织表达 RNA干扰 凋亡 日本对虾 

分 类 号：Q344.1[生物学—遗传学]