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

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%,21.59%and 33.70%respectively.The results showed that MjBcl-2 may play an important role in the immune defense response of M.japonicus to low temperature stress.