日本鳗鲡谷胱甘肽过氧化物酶1和4的克隆、分析和组织表达分布  

Molecular cloning,characterization and tissue distribution of glutathione peroxidase 1 and 4 from Japanese eel,Anguilla japonica

作　　者：刘振兴[1] 柯浩[1] 曹艳林[1,2] 张健騑[1] 林敏[1] 孟轩[1] 

机构地区：[1]广东省农业科学院兽医研究所广东省兽医公共卫生公共实验室,广东广州510640 [2]华南农业大学生命科学学院,广东广州510642

出　　处：《中国水产科学》2010年第3期439-447,共9页Journal of Fishery Sciences of China

基　　金：广东省教育部产学研项目(2006D90204008);东莞市科研发展专项基金项目(2006D055)联合资助

摘　　要：采用RACE技术,克隆了日本鳗鲡(Anguilla japonica)谷胱甘肽过氧化物酶1和4(GPx1、GPx4)基因的完整编码序列(Complete coding sequence,CDS)。GPx1基因全长993bp,5′非编码区(UTR)29bp,CDS573bp,3′UTR372bp,PolyA19bp;第803-898位碱基(位于3′UTR)形成1个硒半胱氨酸插入序列(Selenocysteine insertion sequence,SECIS),协助144-146位密码子TGA编码Sec。GPX4基因全长1048bp,5′UTR115bp,CDS561bp,3′UTR346bp,polyA26bp,第766-863位碱基(位于3′UTR)形成1个SECIS,协助299-301位密码子TGA编码Sec。GPx1包含190个氨基酸,分子量21.4kD,等电点8.04,第21位氨基酸具有1个潜在的N-糖基化位点。GPx4包含186个氨基酸,分子量21.4kD,等电点8.85,第68位和153位氨基酸具有2个潜在的N-糖基化位点。GPx1、GPx4均具有Sec、Trp、Gln和Asn构成的催化四联体。日本鳗鲡与其他脊椎动物相比,GPx1的核苷酸序列一致性为42.7%～60.2%,氨基酸序列一致性为56.4%～80.4%;GPx4的核苷酸序列一致性为46.5%～60.2%,氨基酸序列一致性为59.9%～81.2%。进化分析显示,脊椎动物的GPx1、GPx4分别占据进化树的不同分支。利用Swiss-Model预测了日本鳗鲡GPx1、GPx4单体的3D模型,序列分析显示,GPx1可以形成1个同源四聚体。本研究在克隆日本鳗鲡β-actin基因部分CDS序列的基础上,采用Real-timeRT-PCR方法,检测了日本鳗鲡GPx1、GPx4基因表达,比较了GPx1、GPx4在鳃、皮肤、肌肉、肝、脾、肾、肠组织中的表达变化,发现在鳗鲡的肠、肌肉、肝脏等组织中GPx1、GPx4明显表达,并且GPx1的表达量高于GPx4。Full length cDNAs encoding glutathione peroxidase 1（ GPx1） and glutathione peroxidase 4（ GPx4） of Japanese eel,Anguilla japonica were obtained from liver by a reverse-transcription polymerase chain reaction（ RT-PCR） and rapid amplification of cDNA（ RACE）. The GPx1 is 993 bp,including a coding sequence（ CDS） of 573 bp,a 5 untranslated region of 29 bp,a 3 untranslated region of 372 bp,and a 19 bp poly A tail. The GPx4 is 1 048 bp,including a coding sequence（ CDS） of 561 bp,a 5 untranslated region of 115 bp,a 3 untranslated region of 346 bp,and a 26 bp poly A tail. GPx1 and GPx4 both contain a putative selenocysteine residue which is encoded by the unusual stop codon of TGA,with the selenocysteine insertion sequence（ SECIS） locating in 3 untraslated region. The GPx1 and GPx4 are predicted to separately encode proteins of 190 amino acids and 186 amino acids. The molecular weights of GPx1 and GPx4 are both nearly 21.4 kD. Estimated pI of GPx1 is 8.04 and GPx4’s is 8.85. One N-glycosylation site is found in GPx1 and two in GPx4. GPx1 and GPx4 both possess a classic catalytic tetrad composed of selenocysteine,tryptophan,asparagine and glutamine. The nucleotide identity of GPx1 between Japanese eel and other vertebrate animals is 42.7%-60.2%,and amino acid identity is 56.4%-80.4%. The nucleotide identity of GPx4 between Japanese eel and other vertebrate animals is 46.5%-60.2%,and amino acid identity is 59.9%-81.2%. Phylogenic analysis result shows that GPX1 and GPX4 split into two clusters. The 3D structures are predicted with Swiss-Model software. Sequence analysis result suggests that GPx1 is homotetrameric. This research established the methods for determination of the Japanese eel’s GPx1 and GPx4 relative expression level by using Real-time RT-PCR based on the cloning of partial CDS of β-actin gene. The expression analysis of GPx1 and GPx4 was carried out in different tissues including gill,skin,muscle,liver,spleen,kidney and intestine,and the results indicate GPx1 and GPx4 both show

关 键 词：日本鳗鲡 谷胱甘肽过氧化物酶 REAL-TIME RT-PCR 3D模型 

分 类 号：S91[农业科学—水产科学]