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本研究采用c DNA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c DNA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Poly A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q RT-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NaNO_2胁迫处理后,河川沙塘鳢肝组织Mn-SOD基因的m RNA表达呈先升高后降低的趋势。而鳃组织Mn-SOD基因的m RNA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NaNO_2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。
In this study, we cloned and expressed the Mn-SOD gene of Odontobutis potamophila by rapid amplification of cDNA ends (RACE) and real-time fluorescence quantitative PCR. The results showed that the cloned cDNA sequence of Mn-SOD gene was 1008 bp, including 678 bp open reading frame (ORF), 15 bp 5’UTR and 315 bp 3’UTR , And possesses AATAAA, a typical vertebrate tailed signal and Poly A tail of 31 bp. It is speculated that this sequence encodes a total of 225 amino acids. This gene contains two conserved domains of Sod_Fe_N (28-109) and Sod_Fe_C (116-219). This structure is very similar to other species, indicating that the gene is more conserved in species evolution. The homology comparison between Mn-SOD and known species Mn-SOD showed that the amino acid sequence of Mn-SOD was the highest, 90.3% compared with Oplegnathus fasciatus and Channa striata. The expression of Mn-SOD gene in 8 tissues (kidney, liver, muscle, brain, spleen, gill, eye and heart) and 8 developmental stages (fertilized egg stage, morula stage , Gastrulation stage, neuroblast stage, body-section stage and mouth-split stage, 1 d after operation and 3 d after operation). The expression of Mn-SOD gene was found in all the 8 tissues tested, and the highest expression was found in muscle. The expression of Mn-SOD gene was observed in 8 developmental stages from embryo to larva and highest in morula. After acute NaNO_2 stress, the m RNA expression of Mn-SOD gene in the liver tissue of S. tanguticus increased firstly and then decreased. However, the m RNA expression of Mn-SOD gene in gills decreased first and then increased then decreased. Studies have shown that Mn-SOD is likely to play an important role in counteracting the oxidative damage caused by NaNO 2 stress, which will provide valuable reference for controlling artificial breeding and breeding conditions of Saxophora sp.