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当前位置: 首页 > 文章 > 青虾冷休克蛋白Y-box编码基因的cDNA全长克隆与表达分析 水产学报 2017 (9) 1345-1354
Position: Home > Articles > Molecular cloning and expression analysis of cold shock protein Y-box gene from oriental river pawn(Macrobrachium nipponense) Journal of Fisheries of China 2017 (9) 1345-1354

青虾冷休克蛋白Y-box编码基因的cDNA全长克隆与表达分析

作  者：
孙盛明;傅洪拓;戈贤平;朱健;乔慧;金舒博;张文宜
单  位：
中国水产科学研究院淡水渔业研究中心农业部淡水渔业与种质资源利用重点实验室;南京农业大学无锡渔业学院
关键词：
青虾;冷休克蛋白;低温;低氧;原核表达
摘  要：
为研究冷休克蛋白Y-box基因在青虾应答环境胁迫过程中所起的调控作用,实验应用RACE PCR技术首次克隆了青虾的冷休克蛋白Y-box基因全长c DNA序列,并利用在线软件对其序列特征进行生物信息学分析;采用实时荧光定量PCR技术对其在青虾不同组织及环境胁迫过程中的表达变化特征进行分析。青虾冷休克蛋白Y-box基因c DNA全长1501 bp,包括84 bp的5′末端非翻译区(UTR),876 bp的开放阅读框(ORF),541 bp的3′UTR,开放阅读框编码291个氨基酸。氨基酸相似度比对显示,青虾冷休克蛋白Y-box基因富含高度保守的冷休克结构域。系统进化树分析显示,青虾冷休克蛋白Y-box基因与水蚤等节肢动物冷休克Y-box聚类一支,具有最近的亲缘关系。荧光定量PCR检测显示,冷休克蛋白Y-box基因在青虾不同组织中均有表达,其表达量在肝胰腺组织中最高,使用荧光定量PCR检测青虾冷休克蛋白Y-box基因在低温胁迫和恢复条件下在肝胰腺中的m RNA时空表达情况,结果显示,与对照组相比冷休克蛋白Y-box在肝胰腺中的表达量分别在低温和低氧胁迫3,6和12 h出现了显著上调,而在恢复刺激后其表达量与对照组差异不显著。此外,本实验对Y-box进行了原核表达,为进一步研究Y-box基因的功能奠定了基础。
译  名：
Molecular cloning and expression analysis of cold shock protein Y-box gene from oriental river pawn(Macrobrachium nipponense)
作  者：
SUN Shengming;FU Hongtuo;GE Xianping;ZHU Jian;QIAO Hui;JIN Shubo;ZHANG Wenyi;Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture,Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences;Wuxi Fishery College, Nanjing Agricultural University;
关键词：
Macrobrachium nipponense;;cold shock protein;;low temperature;;hypoxia;;prokaryotic expression
摘  要：
The Y-box protein family is highly conserved nucleic acid binding proteins which are conserved from bacteria to human. In this study, the c DNA encoding cold shock protein Y-box from oriental river pawn Macrobrachium nipponense(Mn Y-box) was cloned by using EST analysis and rapid amplification of c DNA ends(RACE) approach. The full length c DNA of Mn Y-box was 1501 bp, consisting of a 5' untranslated region of 84 bp, a 3' untranslated region of 541 bp, and an open reading frame of 876 bp. The deduced protein had 291 amino acid residues with molecular mass of 31.48 k Da. Based on the protein similarity comparison, the Y-box protein family was very conservative in CSD domain. Phylogenetic tree analysis stated that oriental river prawn has the closest relationship with Daphnia pulex. Quantitative real-time RT-PCR analysis showed that the Mn Y-box gene was expressed in haemocytes, hepatopancreas, muscles, gill, brain, testis, ovary and intestines with the highest level of expression in the hepatopancreas. After environmental low temperature and recovery challenge, the relative expression level of Mn Y-box in hepatopancreas was significantly higher compared to the control group at3 h to 12 h post-low temperature, followed by return to control levels at recovery 12 h. What's more, we constructed prokaryotic expression system and obtained the recombinant proteins of M. nipponense Y-box, which may provide information for further functional study.

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