三孢布拉氏霉乳清酸核苷-5’-单磷酸脱羧酶基因的PCR扩增与序列分析

Cloning and Sequence Analyse of pyrG Gene Encoding Orotidine-5'-monophosphate Decarboxylase in Blakeslea trispora

根据对卷枝毛霉Mucor circinelloides、布氏须霉PPhycomyces blakesleeanu、雪白根霉Rhizopus niveu、少根根霉Rhizo pus arrhizus和微小根毛霉Rhizomucor pusillus的乳清酸核苷-5'-单磷酸脱羧酶基因核酸序列的同源性分析,在第3个外显子内根据卷枝毛霉基因序列设计1对引物,以三孢布拉氏霉Blakeslea trispora基因组DNA为模板进行PCR扩增,得到1个长度约为500 bp的产物。经过同源性分析证明该产物为三孢布拉氏霉乳清酸核苷-5'-单磷酸脱羧酶基因片段。在此基础上,采用抑制性PCR扩增技术,进行染色体步行,分别克隆染色体上相邻的DNA片段,测定了基因的全序列,并对该序列进行了分析。结果表明:在外显子区域核酸序列与卷枝毛霉、少根根霉和微小根毛霉的同源率分别为82％,81％和79％。

Based on the homology analysis of nucleic acid sequence encoding orotidine-5'-monophos-phate decarboxylase from Mucor circinelloides, Phycomyces blakesleeanus, Rhizopus niveus and Rhizo-mucor pusillus, a pair of primers were designed in the third exon. A DNA fragment of about 500 bp in size was amplified from Blakeslea trispora genome by PCR using the pirmers. Homology analysis showed that the product is a part of the pyrG gene encoding orotidine-5'-monphosphate decarboxylase. Then walking upstream and downstream in genomic DNA from the known sequence was proceeded by a suppression PCR . The flanking DNA fragments were amplified and cloned,and the whale gene was se-quenced a ligned. The result indicated that homology of the nucleic acid sequence in the third exon was 82%,81% and 79% to that in M. circinelloides,R. arrbizus and R. pusillus,respectively.