日本囊对虾过氧化氢酶cDNA全长克隆及表达分析

The Full Length cDNA Cloning and Expression Analysis of Catalase from the Kuruma Shrimp(Marsupenaeus japonicus)

采用反转录PCR(reverse transcription PCR,RT-PCR)及cDNA末端快速扩增(rapid-amplification of cDNA ends,RACE)技术,在日本囊对虾(Marsupenaeus japonicus)肝胰腺组织克隆到过氧化氢酶(catalase,CAT)基因cDNA全序列.日本囊对虾CAT基因(MjCAT)cDNA全长1 802bp,开放阅读框(open reading frame,ORF)长1 563bp,编码520个氨基酸,理论等电点为7.26,分子质量为58.9ku.同源建模分析表明,MjCAT蛋白分子结构高度保守.同源性比对后发现,MjCAT基因氨基酸序列与中国明对虾(Fenneropenaeus chinensis)相似性最高(94.2%),其次是凡纳滨对虾(Litopenaeus vannamei)(90.9%).随后的聚类分析表明,其与中国明对虾和凡纳滨对虾聚为一支.荧光定量结果显示,MjCAT基因在检测的多种组织中均有表达,其中以肝胰腺中表达量最高,其次为眼柄、肠、鳃、胃、心脏和血细胞,在肌肉中的表达水平最低.白斑综合征病毒(white spot syndrome virus,WSSV)感染12h后,肝胰腺MjCAT基因的表达量逐渐升高,48h上调至最高的表达水平.研究表明,MjCAT基因在维持机体稳态及抗氧化防御反应过程中发挥着重要的作用.

The catalase gene（MjCAT） from the hepat opancreas of Marsupenaeusjaponicuswas cloned using reverse transcription PCR（RTPCR） and rapid-amplification of cDNA ends（RACE） methods in the present study.The full length cDNA of MjCAT consisted of 1 802 bp with an open reading frame（ORF,1 563 bp）encoded 520 amino acid resides.The molecular mass and isoelectric point of MjCAT protein were predicted to be 58.9 ku and 7.26 respectively.Homology modeling revealed that the potential spatial structure of MjCAT protein was highly conserved.By homologous analysis,the amino acid sequence of MjCAT had the highest similarity （94.2%） with that from Fenneropenaeus chinensis,and followed by Litopenaeus vannamei （90.9 %）.Then phylogenetic analysis indicated that its was first clustered together with F.chinensis and L.vannamei.Real-time quantitative PCR results displayed that MjCAT transcripts could be detected in all the tested tissues,with the highest expression level in the hepatopancreas,then in eyestalk,intestine,gills,stomach,heart and hemocytes,and the lowest in muscle.After white spot syndrome virus （WSSV） infection,the expression level of MjCAT in the hepatopancreas began to increase gradually after 12 h and reached apeak at 48 h.These findings suggested that MjCAT was of pivotal importance for the maintenance of internal homeostasis and antioxidant defense in M.japonicus.