异育银鲫过氧化物还原酶基因的克隆及其表达分析

cDNA Cloning,Characterization and Expression Analysis of Peroxiredoxin Gene in Carassius auratus gibelio

采用反转录PCR和cDNA末端快速扩增技术从异育银鲫Carassius auratus gibelio肝脏中克隆了过氧化物还原酶(Prx)基因的cDNA全长序列。结果显示,异育银鲫过氧化物还原酶(CaPrx)基因cDNA全长1035 bp,开放阅读框长594 bp,编码197个氨基酸。在氨基酸序列的N端和C端各有一个保守的Cys残基,属于2-半胱氨酸类Prxs家族。多序列比对和系统进化树分析表明,异育银鲫与鲫Carassius auratus、青鱼Mylopharyngodon piceus、斑马鱼Danio rerio、犀角金线鲃Sinocyclocheilus rhinocerous和朝鲜鳑鲏Rhodeus uyekii等鱼类的Prx基因具有很高的同源性。实时荧光定量PCR结果显示,CaPrx基因在异育银鲫体内广泛表达,在血细胞和肝脏中表达量最高,在鳃和头肾的表达量较少。在感染鲤疱疹病毒Ⅱ型的个体中,肝脏和脾脏的CaPrx基因表达量显著下降,表明其抗氧化作用因病毒的感染而受到了一定的破坏。

In this study,the full-length cDNA sequence of peroxiredoxin（ Prx） gene was cloned from the liver of Carassius auratus gibelio by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends methods. The structure and speculated function of Prx gene were analyzed. The full-length of CaPrx was comprised of 1 035 nucleotides with a 3＇untranslated region（ UTR） of 366 bp,5＇UTR of 75 bp and an open reading frame of 594 bp,encoding 197 amino acids. The predicted molecular mass and estimated isoelectric point were 21. 83 k D and 5. 93. In addition,there was a conservative Cys in the N-terminal and C-terminal,respectively,and this suggested that the CaPrx is belonging to2-Cys Prxs family. Sequence comparison revealed that the deduced amino acid sequence of CaPrx was highly homologous to the Prx homologs of Mylopharyngodon piceus（ 98%） and Rattus norvegicus（ 81%）. Furthermore,quantitative real-time PCR analysis showed that CaPrx could be detected in various tissues. The highest expression level was detected in hemocyte and liver followed by hemocyte,and little expression of this gene was detected in head kidney,gills and brain. Compared with the diseased fish,the levels of CaPrx was decreased significantly in liver,which indicated that the CaPrx might be involved in immune response against cyprinid herpesvirus Ⅱ infection in C. auratus gibelio.