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张行, 田晓晓, 董文芳, 王茹梦, 潘红春. 中国绿水螅抗坏血酸过氧化物酶基因的克隆、抗体制备及表达分析[J]. 水生生物学报, 2019, 43(2): 305-314. DOI: 10.7541/2019.038
引用本文: 张行, 田晓晓, 董文芳, 王茹梦, 潘红春. 中国绿水螅抗坏血酸过氧化物酶基因的克隆、抗体制备及表达分析[J]. 水生生物学报, 2019, 43(2): 305-314. DOI: 10.7541/2019.038
ZHANG Hang, TIAN Xiao-Xiao, DONG Wen-Fang, WANG Ru-Meng, PAN Hong-Chun. MOLECULAR CLONING, ANTIBODY PREPARATION AND EXPRESSION ANALYSIS OF ASCORBATE PEROXIDASE IN HYDRA SINENSIS[J]. ACTA HYDROBIOLOGICA SINICA, 2019, 43(2): 305-314. DOI: 10.7541/2019.038
Citation: ZHANG Hang, TIAN Xiao-Xiao, DONG Wen-Fang, WANG Ru-Meng, PAN Hong-Chun. MOLECULAR CLONING, ANTIBODY PREPARATION AND EXPRESSION ANALYSIS OF ASCORBATE PEROXIDASE IN HYDRA SINENSIS[J]. ACTA HYDROBIOLOGICA SINICA, 2019, 43(2): 305-314. DOI: 10.7541/2019.038

中国绿水螅抗坏血酸过氧化物酶基因的克隆、抗体制备及表达分析

MOLECULAR CLONING, ANTIBODY PREPARATION AND EXPRESSION ANALYSIS OF ASCORBATE PEROXIDASE IN HYDRA SINENSIS

  • 摘要: 为探讨中国绿水螅(Hydra sinensis)抗坏血酸过氧化物酶(Ascorbate peroxidase, APX)基因的起源及功能, 研究采用RACE方法克隆了中国绿水螅APX基因的全长cDNA序列。该cDNA序列总长1357 bp, 包括5′非编码区107 bp, 3′非编码区146 bp及开放阅读框(Open reading frame, ORF) 1104 bp, 共编码367个氨基酸, 预测蛋白质分子量为40.79 kD。BLAST结果表明中国绿水螅APX蛋白同源序列绝大部分来自植物界; 通过最大似然法(Maximum-likelihood)和贝叶斯分析(Bayesian inference)进行的系统发生分析显示植物界及动物界物种的APX序列各自形成单系群。把APX基因ORF全长序列克隆到原核表达质粒pET-GST中, 重组质粒转化E. coli BL21 (DE3)菌株, IPTG诱导后成功表达重组融合蛋白GST-APX, 再使用纯化的重组蛋白免疫新西兰兔制备多克隆抗体用于APX蛋白的免疫印迹分析(Western blotting assay, WB)。在不同光照时长梯度(光强度2000 lx, 每天分别光照0、4h、8h、12h、16h、20h及24h)下培养中国绿水螅30d, 实时定量PCR (Quantitative real-time PCR, qPCR)及WB检测结果均表明光照时间较长时(每天光照12h以上)绿水螅APX表达呈现一定程度的上调。在长时间光辐射下水螅体内共生绿藻连续进行光合作用所累积的大量活性氧能够扩散到水螅细胞内, 此时水螅体内表达上调的APX可能参与清除其细胞内的活性氧。

     

    Abstract: In order to explore the origin and physiological function of ascorbate peroxidase (APX) gene of Hydra sinensis, a full-length cDNA of APX gene of H. sinensis was isolated by rapid amplification of cDNA ends (RACE) technique. The full-length cDNA of APX gene was 1357 bp, containing 107 bp 5′UTR (Untranslated region), 146 bp 3′UTR and a 1104 bp open reading frame (ORF) which encodes a polypeptide of 367 amino-acid residues with a molecular weight of 40.79 kD. BLAST (Basic Local Alignmen Search Tool) showed that most of homologous protein sequences of APX in H.sinensis belonged to the plant kingdom. Phylogenetic results by Maximum Likelihood (ML) method and Bayesian analyses revealed that homologous sequences of APX from plant and animal kingdom each formed a single cluster. The ORF of APX gene was subcloned into plasmid pET-GST and transferred into Escherichia coli BL21 (DE3). The recombinant GST-APX fusion protein mainly expressed in the form of soluble were immunized New Zealand rabbits get the polyclonal antibody against APX for Western blotting assay (WB). H. sinensis was cultured (0, 4, 8, 12, 16, 20 and 24 hours per day with illumination intensity 2000 lx) for 30 days, which up-regulated APX expression under longer illumination time (more than 16 hours per day). Due to continuous photosynthetic activity, a large number of reactive oxygen species (ROS) accumulating in symbiotic green algae could spread to the host cell (hydra cell), and the upregulation of APX expression in H. sinensis may mediate the removal of intracellular ROS.

     

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