巴西龟古洛糖酸内酯氧化酶基因全长cDNA的克隆及其组织表达特异性分析  

Full length cDNA cloning and tissue expression specificity of L-gulonolactone oxidase gene in Red-ear turtle (Trachemys scripta)

作　　者：龚勋[1,2] 牛翠娟[1] 

机构地区：[1]北京师范大学生命科学学院/生物多样性与生态工程教育部重点实验室,北京100875 [2]毕节学院地理与生命科学学院,贵州毕节551700

出　　处：《广东农业科学》2013年第7期130-135,共6页Guangdong Agricultural Sciences

基　　金：高等学校博士学科点专项科研基金(20100003110022);贵州省科学技术基金(黔科合J字[2013]2007号)

摘　　要：采用SMARTFM RACE技术从巴西龟（Trachemysscripta）的肾脏组织中克隆了L-古洛糖酸内酯氧化酶（GLO）基因全长cDNA序列，其长度为1875bp，5＇UTR251bp，3＇UTR301bp，ORF1323bp，编码440个氨基酸，分子量50．5ku，理论等电点为6．51。氨基酸序列同源性分析结果表明：巴西龟与中华鳖同源性最高，相似度和一致性分别为95．7％和88％；其次为鸟类（红原鸡）．分别为94．8％和85．9％；与高等哺乳类相似度和一致性达92．7％～87．4％和84．1％～78．4％；与鱼类较低，分别为88．9％～82．7％和73．9％～64．8％；与鸭嘴兽最低，分别为72．4％和64．2％。分子系统进化结果也表明巴西龟与中华鳖及鸟类（红原鸡）的GLO分子进化关系最近。从mRNA、蛋白及酶活性表达水平对巴西龟的6种不同组织（脑、脾、肝、肾、肌肉和肠）进行了组织表达差异分析，结果发现巴西龟GLOmRNA在肾脏和肌肉组织中均有转录，但Western—blot结果表明GLO蛋白只在龟的肾脏中表达，HPLC结果也显示仅肾脏中存在极显著的GLO酶活性。以上结果表明巴西龟GLO基因在进化中具有高度保守性，其表达具有组织特异性。In this paper, we cloned the complementary DNA （cDNA） for the GLO gene in red-ear turtle Trachemys scripta. The T. scripta GLO cDNA was 1 875 bp long and consisted of a 251 bp 5＂-untranslated region （UTR）, a 301 bp 3＇-UTR, and a 1 323 bp open reading frame （ORF） encoding a 440-amino-acid protein with 50.5 ku predicted molecular mass and 6.51 theoretical isoelectric point （pI）. Using MatGAT software, the amino acid sequence for T. scripta GLO was analyzed against other known GLO sequences, and results revealed highest similarity （95.7%） and identity （88%） with soft-shelled turtle Pelodiscus sinensis. Phylogenetie analysis also revealed that the T. scripta GLO amino acid sequence clustered together with soft-shelled turtle and red jungle fowl. RT-PCR analysis in 6 kinds of tissues indicated that the T. scripta GLO mRNA expressed in kidney and muscle and did not expressed in brain, spleen, liver and gut. However, western blotting results indicated that GLO protein was only expressed in kidney. GLO enzyme activity was also measured by the method of high performance liquid chromatography （HPLC） in six tissues of T. scripta. The results showed that GLO activity was markedly higher in kidney tissue. Therefore, T. scripta GLO gene was of high conservation in the course of vertebrate evolution. The expression level of T. scripta GLO mRNA was pattieular to tissues. Via both western-blot and enzyme activity analysis, we determined that GLO protein expression in red-ear turtle was kidney specific.

关 键 词：巴西龟 GLO 基因克隆 组织表达 

分 类 号：Q959.63[生物学—动物学]