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河川沙塘鳢(Odontobutis potamophila) Mn-SOD基因的克隆及表达分析
张宏叶1, 于兴达1, 王佩佩1, 贾秀琪1, 李欣茹1, 曹全全1, 尹绍武1, 陈树桥2, 周国勤2
1.南京师范大学生命科学学院 江苏省生物多样性与生物技术重点实验室 南京 210023;2.南京市水产科学研究所 南京 210036
摘要:
本研究采用cDNA末端快速扩增(RACE)和实时荧光定量PCR等技术,对河川沙塘鳢(Odontobutis potamophila)的Mn-SOD基因进行了克隆和表达模式分析。结果显示,克隆得到的河川沙塘鳢Mn-SOD基因的cDNA序列全长为1008 bp,包括678 bp的开放阅读框(ORF),15 bp的5¢UTR区和315 bp的3¢UTR区,并且具有脊椎动物典型的加尾信号AATAAA和31 bp的PolyA尾巴,推测该序列共编码225个氨基酸。该基因包含Sod_Fe_N (28-109)与Sod_Fe_C (116-219)2个保守的结构域,此结构与其他物种极为相似,表明该基因在物种进化中比较保守。与已知物种Mn-SOD进行同源性比对发现,河川沙塘鳢Mn-SOD氨基酸序列与条石鲷(Oplegnathus fasciatus)和线鳢(Channa striata)相似性最高,均为90.3%。采用qRT-PCR技术检测了Mn-SOD基因在河川沙塘鳢8个组织(肾、肝、肌肉、脑、脾、鳃、眼、心脏)和8个发育时期(受精卵期、桑椹胚期、原肠胚期、神经胚期、体节期、口裂期、出膜后1 d、出膜后3 d)的表达情况。在检测的8个组织中都有Mn-SOD基因的表达,肌肉中的表达量最高;胚胎到仔鱼的8个发育时期都有Mn-SOD基因的表达,桑椹胚期表达量最高。在急性NaNO2胁迫处理后,河川沙塘鳢肝组织Mn-SOD基因的mRNA表达呈先升高后降低的趋势。而鳃组织Mn-SOD基因的mRNA表达呈先降低后升高再降低的趋势。研究表明,Mn-SOD很可能在对抗NaNO2胁迫引起的氧化损伤中起重要作用,这将为控制河川沙塘鳢的人工育苗及养殖条件提供有价值的参考资料。
关键词:  河川沙塘鳢  Mn-SOD基因  基因克隆  mRNA表达量
DOI:10.11758/yykxjz.20160725001
分类号:
基金项目:
Cloning and Expression Analysis of Mn-SOD Gene of Odontobutis potamophila
ZHANG Hongye1, YU Xingda1, WANG Peipei1, JIA Xiuqi1, LI Xinru1, CAO Quanquan1, YIN Shaowu1, CHEN Shuqiao2, ZHOU Gouqin2
1.College of Life Sciences, Key Laboratory of Biodiversity and Biotechnology of Jiangsu Province, Nanjing Normal University, Nanjing 210023;2.Institute of Fisheries Science of Nanjing city, Nanjing 210036
Abstract:
Superoxide dismutase (SOD), the first barrier of antioxidation, protects cells through the removal of excessive superoxide. However, little is known about the function of SOD genes in Odontobutis potamophila. In the present study, the rapid amplification of cDNA ends (RACE) approach and quantitative real-time PCR were used to clone the full-length cDNA of Mn-SOD and analyze its expression patterns. The full length of cDNA Mn-SOD was 1008 bp, including an open reading frame (ORF) of 678 bp, a 5¢-untranslated regions (UTR) of 15 bp and a 3¢UTR of 315 bp. Each 3¢UTR possessed a polyadenylation signal sequences of AATAAA and a poly A tail of 31 bp, a phenomenon that vertebrate usually has. It was speculated that the sequence encoded 225 amino acids. The Mn-SOD genes contains two conserved domains: the Sod_Fe_N (28-109) and Sod_Fe_C (116-219). The structure was similar with other species, which showed that the gene was conserved in the evolution. The homologous comparison with other species revealed that the Mn-SOD had the highest sequence identity with Oplegnathus fasciatus and Channa striatawhich, about 90.3 %. Quantitative real-time PCR was used to quantify the tissue-specific expression of Mn-SOD gene. Predominant expression of Mn-SOD was detected in muscle. Different developmental stages from embryo to larva had Mn-SOD gene expressed. Morula stage had the highest expression level. In addition, the expression levels of Mn-SOD mRNA in liver increased initially and then declined after the exposure of NaNO2. In gill, the mRNA expression levels of Mn-SOD fluctuated significantly. The findings of this study suggested that the Mn-SOD may play an important role in defending oxidative stress and cellular damage induced by nitrite, by detoxifying harmful reactive oxygen. Our study provides valuable reference to optimize the artificial breeding and rearing conditions of O.potamophila.
Key words:  Odontobutis potamophila  Mn-SOD gene  Gene cloning  mRNA expression level