苹果L-半乳糖脱氢酶基因 cDNA全长的克隆与序列分析

L-半乳糖脱氢酶(L-Galactose dehydrogenase,GalDH)是维生素C合成的L-半乳糖途径中,催化L-半乳糖生成L-半乳糖内酯的关键酶。根据GenBank中登录的的GalDH cDNA序列设计1对扩增引物,以嘎拉苹果叶片为材料,采用RT-PCR法扩增出GalDH cDNA全长。将获得的基因片段克隆到pMD18-T载体上,转入大肠杆菌DH5α筛选阳性克隆,经酶切和PCR鉴定,并对插入片段进行序列分析,结果表明,本试验获得的cDNA片段长为1 111 bp,是苹果GalDH cDNA全长。

Cloning and sequence analysis of full-length cDNA encoding L-Galactose dehydrogenase from apple

L-Galactose dehydrogenase (L-GalDH) is the key enzyme of ascorbate biosynthesis and oxidizing L-Gal to L-galactono-1,4-lactone. Full-length cDNA encoding L-GalDH was cloned from Royal Gala apple(Malus Pumila Mill cv. Royal Gala) leaves by RT-PCR method according to the homologous gene in GenBank. Then the target fragment was purified from agarose gels and integrated into pMD18T cloning vector. After being transformed into E. coli . DH 5a. tested by restriction endonuclease digestion and PC , the screened positive clone was sequenced. Sequencing analysis shows that the length of the cDNA of apple GalDH is 1 111 bp,which is the full-length cDNA oi GalDH.