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

  • 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。

     

    Abstract: 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.

     

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