家蚕磷酸吡哆醇氧化酶cDNA克隆、鉴定及基因结构分析  

Cloning,Identification and Genomic Organization of the cDNA Encoding Pyridoxine 5’-phosphate Oxidase in Bombyx mori

作　　者：张剑韵[1] 石瑞君[1] 黄硕豪[1] 黄龙全[2] 

机构地区：[1]安徽农业大学生命科学学院,合肥230036 [2]安徽农业大学茶与食品科技学院,合肥230036

出　　处：《中国农业科学》2008年第11期3784-3792,共9页Scientia Agricultura Sinica

基　　金：安徽省教育厅自然科学研究项目(2003KJ126);国家自然科学基金资助项目(30870338)

摘　　要：【目的】了解家蚕维生素B6关键代谢酶磷酸吡哆醇氧化酶(PNPO)的基因结构。【方法】利用生物信息学原理和PCR方法,克隆出编码家蚕(Bombyx mori)PNPO的cDNA(GenBank登录号:DQ452398);采用T7启动子/T7RNA聚合酶原核表达系统对cDNA进行体外表达,并通过酶活检测进行表达产物的功能鉴定;依据家蚕基因组数据库信息和得到的cDNA,对家蚕PNPO基因结构进行分析。【结果】克隆到的cDNA含有771bp的完整可读框,编码一条分子量为29.86kD、含257个氨基酸残基的蛋白质。序列比对显示此蛋白质与人类PNPO具有51.44%的同源性,包含PNPO家族共有的保守序列,但在人和大肠杆菌PNPO中具有重要作用的几个氨基酸残基在此蛋白质中被替换。在以磷酸吡哆醇(PNP)为底物时,表达产物Pro-PNPO的酶活力约为17nmol·min-1·mg-1。家蚕PNPO基因包含5个外显子和4个内含子,跨越3.5kbDNA序列,所有外显子/内含子交接点都遵从gt/ag剪接规则,基因的5’端启动子调控区发现有TATA-box和CAAT-box保守基序。【结论】获得家蚕PNPO基因,可利用该基因在分子水平上研究家蚕的VB6代谢特征,弄清PNPO家族的特征及其在生物进化过程中的演变规律。[Objective] Pyridoxine 5＇-phosphate oxidase （PNPO） （EC 1.4.3.5） is a key enzyme that catalyzes the vitamin B6 （VB6） metabolism. The objective of this paper is to study the gene structure of PNPO in Bombyx mori. [ Method] A cDNA encoding PNPO was cloned from Bombyx mori using the PCR method （GenBank accession number： DQ452398）. The cDNA cloned was expressed in E. coli using the T7 promoter/T7 RNA polymerase expression system. According to genomic database ofBombyx mori and the cDNA of PNPO, the gene structure ofB. mori PNPO was determined. [Result] The cDNA of PNPO cloned from Bombyx mori has an 771 bp open reading frame and encodes a protein of 257 amino acid residues with a molecular mass of 29.86 kD. The amino acid sequence shares 51.44% identity with that of human PNPO, and it also contains signature conserved motifs of the PNPO family. However, several conserved amino acid residues in human and E.coli PNPO, which play important roles in the enzyme, are changed in the protein. The crude extracts containing the expressed product of PNPO-cDNA have strong enzymatic activity of PNPO with a value of 17 nmol.min^-1.mg^-1 The gene of B. mori PNPO is composed of five exons and four introns, and spans approximately 3.5 kb. All exon/intron junctions of the gene contain the gt/ag consensus splicing site. The TATA-Iike box and CAAT-like box is found in regulatory region ofPNPO-gene ofBombyx mori. [Conclusion] The cloning ofPNPO-gene ofBombyx mori will not only facilitate further study on VB6 metabolic mechanism of Bombyx mori, but also beneficial to the understanding of the characteristics and evolution of the PNPO family.

关 键 词：家蚕 磷酸吡哆醇氧化酶 CDNA克隆 酶活力 基因结构分析 

分 类 号：S881[农业科学—特种经济动物饲养] Q78[农业科学—畜牧兽医]