宽体沙鳅(Botia reevesae)热休克蛋白70基因的克隆及表达分析  

THE CLONING AND EXPRESSION OF HEAT SHOCK PROTEIN 70 cDNA IN BOTIA REEVESAE

作　　者：覃川杰[1] 顾顺樟 赵大显[3] 陈立侨[4] 李二超[4] 齐泽民[1] 

机构地区：[1]内江师范学院生命科学学院,内江641000 [2]上海机场出入境检验检疫局,上海201207 [3]南昌大学生命科学与食品工程学院,南昌330031 [4]华东师范大学生命科学学院,上海200062

出　　处：《海洋与湖沼》2013年第6期1584-1591,共8页Oceanologia Et Limnologia Sinica

基　　金：四川省教育厅项目,11ZB025号;四川省科技厅项目,2011NZ0075号;国家科技支撑计划课题资助项目,2012BAD25B03号;国家自然科学基金项目,31260642号;江西省自然(青年)科学基金,20132BAB214015号;中国博士后科学基金第六批特别资助,2013T60650号;内江师范学院大学生科研立项,13NSD-77号

摘　　要：为深入分析热休克蛋白响应胁迫的分子机制,实验以宽体沙鳅(Botia reevesae)为研究对象,利用同源克隆和cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术克隆得到宽体沙鳅热休克蛋白70(heat shock protein,BR-HSP70)的cDNA全长。结果发现,BR-HSP70 cDNA全长为2371bp,包含1947bp的开放阅读框(opening reading frame,ORF),102bp 5′-非编码区(untranslated region,UTR)和322bp 3′-UTR等。通过序列同源性比对发现,BR-HSP70 cDNA与团头鲂(Megalobrama amblycephala)和猪(Sus scrofa)的同源性分别为98%及83%,且ORF编码的649个氨基酸中含有HSP70家族的家族信号标签、N-糖基化位点及EEVD等保守区域。结果表明,克隆的cDNA为宽体沙鳅HSP70基因。实时荧光定量PCR分析发现,氨氮胁迫和嗜水气单胞菌(Aeromonas hydrophila)侵染均会显著上调宽体沙鳅鳃、肝脏及肾脏HSP70 mRNA的表达(P<0.05),表明HSP70基因在宽体沙鳅应对环境胁迫中发挥了重要的抗应激作用。We investigated the effect of pathogenic bacterial challenge and acute sublethal ammonia-N exposure on heat shock protein 70 （HSP70） expression in Botia reevesae. Results show that the HSP70 cDNA in B. reevesaeis 2371bp, containing an open reading frame at 1947bp, a 102bp 5＇-untranslated region （UTR）, a 322bp 3＇-UTR, and five common putative functional domains. By sequence comparisons, we found that the deduced amino acid sequence of the HSP70 had an overall identity of 98%, and 83% to that of Megalobrama amblycephala andSus scrofa, respectively. Real time quantitative PCR analysis showed the HSP70 mRNA expression levels was significantly （P〈0.05） up-regulated in gill, liver, spleen, and kidney by Aeromonas hydrophilachallenge or under ammonia-N exposure. It is believed that B. reevesae HSP70 is involved in the resistance to pathogenic bacteria stress.

关 键 词：宽体沙鳅 热休克蛋白70 氨氮 嗜水气单胞菌 胁迫 

分 类 号：Q953[生物学—动物学]