红罗非鱼Na+-K+-ATPase α基因的克隆及其在不同盐度条件下mRNA表达差异

生物技术通报 ›› 2014, Vol. 0 ›› Issue (3): 137-145.

红罗非鱼Na⁺-K⁺-ATPase α基因的克隆及其在不同盐度条件下mRNA表达差异

王忠良^1，2, 张健东^1，2, 黄建盛^1，2, 汤保贵^1，2, 周晖^1，2, 施钢^1，2, 潘传豪^1，2, 吴灶和^3，4, 陈刚^1，2

(1. 广东海洋大学水产学院，湛江 524088；2.广东省普通高校南海水产经济动物增养殖重点实验室，湛江 524088；3. 广东省水产经济动物病原生物学及流行病学重点实验室，湛江 524088；4. 仲恺农业工程学院，广州 510225)

收稿日期:2013-09-24 出版日期:2014-03-29 发布日期:2014-03-31
作者简介:王忠良，男，博士，讲师，研究方向:水产经济动物增养殖；E-mail:leong2006@126.com
基金资助:
广东省教育厅育苗项目(B12123)，广东省科技厅专业镇中小微企业服务平台建设项目(2012B091400016)

Cloning and Expression Analysis of Na⁺-K⁺-ATPase α in Red Tilapia(Oreochromis sp.)at Different Water Salinities

Wang Zhongliang^1，2, Zhang Jiandong^1，2, Huang Jiansheng^1，2, Tang Baogui^1，2, Zhou Hui^1，2, Shi Gang^1，2, Pan Chuanhao^1，2, Wu Zaohe^3，4, Chen Gang^1，2

(1. Fisheries College, Guangdong Ocean University，Zhanjiang 524088；2. Key Laboratory of Aquaculture in South China Sea for Aquatic Economic Animal，Regular High Education Institute of Guangdong Province，Zhanjiang 524088；3.Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals，Zhanjiang 524088；4. Zhongkai University of Agriculture and Engineering，Guangzhou 510225)

Received:2013-09-24 Published:2014-03-29 Online:2014-03-31

摘要/Abstract

摘要： 为获知红罗非鱼(Oreochromis sp .)Na⁺-K⁺-ATPase α基因的全长分子结构及其在不同盐度条件下的表达情况，采用同源克隆及cDNA末端快速扩增(RACE-PCR)方法，首次在红罗非鱼鳃组织中克隆到了全长为3 379 bp的Na ⁺-K ⁺-ATPase α基因全长cDNA序列，该序列包含3 072 bp的开放阅读框(ORF)，143 bp的5'末端非编码区(UTR)和164 bp的3'末端非编码区(UTR)，编码1023个氨基酸，预测分子量为112.5 kD，理论等电点为5.26。BLAST分析显示红罗非鱼Na ⁺-K ⁺-ATPase α基因编码的氨基酸序列与其它已知物种相应基因编码的氨基酸序列的同源性达到97%-99%；系统进化分析显示，红罗非鱼Na ⁺-K ⁺-ATPase α亚基与萨罗罗非鱼(Sarotherodon melanotheron)和莫桑比克罗非鱼(Oreochromis mossambicus)亲缘关系较近。应用Real-time PCR技术，以β- actin基因为内参，对不同盐度(0、15、25、32 g/L)条件下鳃组织中Na ⁺-K ⁺-ATPase α基因的表达情况进行了比较分析，结果表明，当盐度为25 g/L时，Na ⁺-K ⁺-ATPase α基因的表达量达到峰值，而盐度升至32 g/L时，其表达量呈下降趋势；各盐度处理组中，养殖12 h后Na ⁺-K ⁺-ATPase α基因的mRNA表达量显著上升(P<0.05)并达到最高；随着养殖时间的延长，24 h后该基因mRNA 表达量开始降低，但仍然显著高于对照组的表达量值(P<0.05)。

关键词: 红罗非鱼 Na⁺-K⁺-ATPase, α基因, cDNA末端快速扩增, 实时荧光定量PCR

Abstract: The full-length cDNA of red tilapia(Oreochromis sp.)Na ⁺-K ⁺-ATPase α was cloned using homological cloning and rapid amplification of cDNA ends(RACE)methods. Results showed that the full-length cDNA was 3 379 bp, containing a 5'untranslated region(5'-UTR)of 143 bp, a 3'-UTR of 164 bp and open reading frame of 3 072 bp bp encoding 1 023 amino acids with an estimated molecular weight of 112.5 kD and an estimated isoelectric point of 5.26. BLAST analysis revealed that the Na ⁺-K ⁺-ATPase α of red tilapia shared a high homology of 97%-99% with the corresponding proteins of other know species, respectively. Phylogenetic analysis showed that Na ⁺-K ⁺-ATPase α of red tilapia shared closest relationship with the corresponding proteins of Oreochromis mossambicus and Sarotherodon melanotheron, respectively. The expression pattern of Na ⁺-K ⁺-ATPase α at different water salinity was analyzed by fluorescent quantitative real-time PCR technology, and results showed that the expression of Na ⁺-K ⁺-ATPase α reached the highest level at 25, and fell to a relatively lower level at 32. In the same salinities, the expression of Na ⁺-K ⁺-ATPase α mRNA increased significantly(P<0.05)during the adaptation phase(12 h), and in regulatory phase(after 24 h)the amounts of Na ⁺-K ⁺-ATPase α mRNA fell to a relatively lower level, while the expression levels were still higher than that of fish in control group(P<0.05).

Key words: Red tilapia(Oreochromis sp .), Na⁺-K⁺-ATPase, α Rapid amplification of cDNA ends(RACE), Fluorescent quantitative real-time, PCR

王忠良, 张健东, 黄建盛, 汤保贵, 周晖, 施钢, 潘传豪, 吴灶和, 陈刚. 红罗非鱼Na⁺-K⁺-ATPase α基因的克隆及其在不同盐度条件下mRNA表达差异[J]. 生物技术通报, 2014, 0(3): 137-145.

Wang Zhongliang, Zhang Jiandong, Huang Jiansheng, Tang Baogui, Zhou Hui, Shi Gang, Pan Chuanhao, Wu Zaohe, Chen Gang. Cloning and Expression Analysis of Na⁺-K⁺-ATPase α in Red Tilapia(Oreochromis sp.)at Different Water Salinities[J]. Biotechnology Bulletin, 2014, 0(3): 137-145.

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