为研究高效产氢菌株成团泛菌(Pantoea agglomerans)BH-18中依赖型磷酸甘油酸变位酶(cofactor-dependent phosphoglycerate mutase, dPGM)与产氢之间的关系,本研究根据GenBank上已登录的肠杆菌中编码 dPGM的基因序列设计一对引物,从细菌基因组 DNA中克隆得到编码 dPGM基因的完整开放阅读框,其长度为753 bp,编码250 aa。采用BLAST对其与NCBI GenBank中的核苷酸序列进行比较分析,结果表明该基因保守性相对较高,与肠杆菌科众多菌株中的 dPGM 基因相似性达100%。采用Bioedit和Mega4软件构建NJ系统进化树,结果表明成团泛菌BH-18的dPGM氨基酸序列与Enterobacter asburiae的dPGM聚为一类,而与成团泛菌属中其他菌株的该蛋白关系较远, dPGM的氨基酸序列在属内不保守。最后,根据已获得的基因序列设计引物,采用RT-PCR的方法分析了成团泛菌BH-18产氢过程中dPGM基因的转录情况,结果表明dPGM基因的转录与产氢呈正相关,依赖型磷酸甘油酸变位酶是产氢过程中的关键酶。%In order to study the relationship between high hydrogen-production and cofactor-dependent phosphoglycerate mutase (dPGM) in Pantoea agglomerans BH-18, a set of primers were designed based on the dPGM-encoding gene sequences of Enterobacteriaceae which have accession number on GenBank. And the dPGM-encoding gene was obtained by PCR amplification from the bacterial genomic DNA. A complete open read-ing frame of the gene was 753 bp and encoded 250 aa. The results of BLAST analysis in GenBank indicated that the nucleotide sequence of the gene was highly conserved, and the similarity to many strains of Enterobacteriaceae reached 100%. Meanwhile, The NJ phylogenetic tree was constructed by the software of Bioedit and Mega4, and the results indicated that the amino acid sequences of dPGM in BH-18 and Enterobacter asburiae belonged to the same class, whereas it was distant from the protein of the other Pantoea sp. The results suggested the amino acid sequence was not conserved in genus. Then another set of primers were designed according to the dPGM-encoding gene sequence of strain BH-18. The transcription changes of dPGM-encoding gene were analyzed by the method of RT-PCR in the process of hydrogen production. The results demonstrated that the transcription of dPGM gene was positively correlated with hydrogen production, which suggested that dPGM was a key enzyme in hydrogen pro-duction.
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