日本沼虾含硒谷胱甘肽过氧化物酶全长克隆及表达分析

FULL-LENGTH cDNA CLONING AND EXPRESSION ANALYSIS OF SELENIUM DEPENDENT GLUTATHIONE PEROXIDASE FROM MACROBRACHIUM NIPPONENSE

为探讨日本沼虾(Macrobrachium nipponense)的免疫机制及含硒谷胱甘肽过氧化物酶(Se-GPx)在甲壳动物解毒和免疫应激反应中的作用,本文采用RACE法克隆了日本沼虾Se-GPx cDNA全长,其cDNA全长为908 bp,5′-UTR的长度为91 bp,3′-UTR长为256 bp,包括1个保守的硒代半胱氨酸插入序列(SECIS)和1个polyA尾,开放阅读框长度为561 bp,编码由186个氨基酸组成的多肽,其中,第39个氨基酸为TGA编码的硒代半胱氨酸。同源性和相似性分析显示日本沼虾的Se-GPx在甲壳动物中与罗氏沼虾相似度最高,在脊椎动物中与GPx1和GPx2家族的相似度比较高。日本沼虾Se-GPx基因在血细胞、肝胰腺、肌肉、卵巢、表皮以及大颚器官中都有表达,尤其在血细胞中表达量相对较高。用嗜水气单胞菌(Aeromonas hydrophila)刺激后3h和6h,血细胞Se-GPx基因表达量显著升高。结果表明,日本沼虾Se-GPx作为一种重要的抗氧化酶,在维护组织正常功能方面及免疫应激反应中发挥重要作用。

Abstract： Reactive oxygen species （ROS） produced in cells contribute to the pathogenesis of several diseases. Particu larly large amounts are produced in inflamed tissues, where they are released by activated macrophages during respira tory bursts that are a defense mechanism against microbial infection. But ROS may be a double-edged sword, its over-expression will cause cellular damage and immune dysfunction. To maintain suitable low levels of ROS, aerobic organisms including crustacean are equipped with an elaborate enzymatic antioxidant defense system, in which glu- tathione peroxidase （GPx）, a key antioxidant enzyme, may protect biomembranes and other cellular components from oxidative damage by catalyzing the reduction of variety of ROS. Investing the GPx gene expression in bacterial chal lenge can help us to understand the defense mechanisms of crustacean. GPx can be classed into two types： the non selenium-dependent GPx （non-Se-GPx） and the selenium-dependent GPx （Se-GPx）. For Se-GPx, four Se-GPx isozymes have been further identified according to cellular location and substrate specificity in vertebrate, named cellular GPx （GPxl）, gastrointestinal GPx （GPx2）, plasma GPx （GPx3） and phospholipid hydroperoxide GPx （GPx4）. In this study, the commercially important freshwater shrimp Macrobrackium nipponense, obtained from the Yuan yang Huangsi farm with an average length of （4.5±0.5） cm, were used. Firstly, the total RNA derived from different tissues was extracted, and then the quality of RNA was checked by agarose gel eleetrophoresis. The full-length eDNA sequence of seleniumdependent glutathione peroxidase （Se-GPx） from M. nipponense was cloned using the reverse transcription polymerase chain reaction （RT-PCR） and rapid amplification of eDNA ends （RACE）. The immune chal- lenge test was carried out by injecting of Aeromonas hydrophila into abdominal segment of each shrimp at a dose of 20 μL （5.0x 106 cells/mL）, respectively. The result showed that the length of Se-GPx gene eDNA was 908 bp, including a 91 bp of 5＇-untranslated region （UTR）, a 256 bp of 3＇-UTR with one selenocysteine insertion sequence （SECIS）, and a polyadenylation signal （AATAAA） with 11 bp upstream of the PolyA tail. The open reading frame （ORF） was 561 bp, encoding a peptide of 186 amino acids with an estimated molecular mass of 21.2 kD and a theoretical isoeletric point of 6.74. The putative Se-GPx amino acid sequence contained a selenocysteine （See） residue which was encoded by the unusual stop codon TGA, a GPx signature motif 63LAFPCNQFT70and an active site motif 151WNFEKF156. The putative N-glycosylation site 75NNT77 and 107NGS109 were observed in the Se-GPx amino acid sequence. Homology analysis of the deduced amino acid sequence of the Se-GPx from M. nipponense with other known species revealed that the Se-GPx was the most similar to Macrobrachium rosenbergii in crustacean, and the Se-GPx was more similar to GPxl and GPx2 than GPx3 and GPx4 in vertebrate. The Se-GPx gene was expressed in many tissues such as haemocyte, hepatopancreas, muscle, ovary, epidermis and mandibular organ, the level of the Se-GPx gene was the highest in haemocyte and higher in hepatopanereas and ovary comparing to other tissues. The immune challenge test revealed that the expression level of the Se-GPx gene in haemocyte was significantly up-regulated （P〈0.05） at 3h and 6h after bacterial challenge, indicating that the Se-GPx was involved in the crustacean immune system.