家蚕吡哆醛激酶cDNA的克隆、表达和基因结构分析.pdfVIP

昆 虫 学 报 Acta Entomologica Sinica，February 2008，51(2)：113—119 ISSN 0454—6296 家蚕吡哆醛激酶cDNA的克隆、表达和基因结构分析 张剑韵 ，石瑞君 ，黄龙全 ’ (i．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) 摘要：吡哆醛激酶(pyfidoxal kinase，PLK，EC2．7．1．35)是维生素取关键代谢酶，其cDNA的克隆在昆虫类还未见报 道。利用生物信息学原理和使用PCR方法，克隆出编码家蚕 Bombyx mori吡哆醛激酶的 cDNA(GenBank登录号 DQ452397)，体外原核表达成功，并对表达粗提产物进行了酶活检测。克隆到的cDNA含有一894 bp的完整可读框， 编码一条分子量为33．1 kD，含 298个氨基酸残基的蛋白质。序列比对显示此蛋白质与人类吡哆醛激酶具有 52．84％的同一性．包含吡哆醛激酶家族共有的特征保守序列，但比哺乳动物和植物克隆到的吡哆醛激酶均少 10多 个氨基酸残基，几个有关键功能且在哺乳动物和植物中均保守的氨基酸残基在此蛋白中被替换。依据家蚕基因组 数据库信息和PLK的cDNA，家蚕PLK基因包含5个外显子和4个内含子，跨越10 kb DNA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。 关键词：家蚕；吡哆醛激酶；cDNA克隆；表达；基因结构分析 中图分类号：$881．2 文献标识码：A 文章编号：0454—6296(2008)02—0l13—07 Cloning， expression and genomic organization of the cDNA encoding pyridoxal kinase in Bombyx mori ZHANG Jian．Yun。，SHI Rui—Jun。，HUANG Long—Quan ’ (1．College of Life Sciences，Anhui Agricultural University，Hefei 230036，China；2．College of Tea＆ Food Science，An ui Agricultural University，Hefei 230036，China) Abstract：Pyridoxal kinase(PLK，EC2．7．1．35)is a key enzyme catalyzes the vitamin B6 metabolism．In this paper．a cDNA encoding PLK was cloned from Bombyx mori using the PCR method．卟e cDNA(GenBank accession number：DQ452397)has an 894 bp open reading frame and encodes a protein of 298 amino acid residues with a molecular mass of 33．1 kD．The cDNA cloned was expressed successfully in Escherichia coli using the T7 promoter／T7 RNA polymerase expression system，and the crude extracts containing the expressed product were found to have strong PLK enzymatic activity．The amino acid sequence shares 52．84％ identity with that of human PLK，and it also contains signature conserved motifs of the PLK family．However，the protein is 10 or more amino acid shorter than the PLKs from