家蚕吡哆醛激酶cDNA的克隆、表达和基因结构分析

Cloning,expression and genomic organization of the cDNA encoding pyridoxal kinase in Bombyx mori

吡哆醛激酶(pyridoxal kinase,PLK,EC2.7.1.35)是维生素B6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含有一894bp的完整可读框,编码一条分子量为33.1kD,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10kb 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。

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. The 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 mammals and plants, and several amino acid residues conserved in the PLKs from mammals and plants are changed in the protein. According to genomic database of B. mor/ and the cDNAs of PLK, the gene organization of PLK was determined. The B. mor/ PLK gene is composed of five exons and four introns, and spans approximately 10 kb. All exon/intron junctions of PLK gene of B. mor/ contain the gt/ag consensus splicing site. The TATA-like box and CAAT-like box is found in regulatory regions of PLK gene. This is the first identification of a gene encoding PLK in insects.