异养硝化菌Alcaligenes faecalis NR氨单加氧酶PCR扩增及生物信息学分析

PCR Amplification and Bioinformatics Analysis of AMO from a Heterotrophic Nitrifier,Alcaligenes faecalis NR

氨单加氧酶(AMO)是异养硝化过程中起催化作用的关键酶之一,其相应的基因为amo。本研究根据NCBI数据库中amo基因序列设计引物,成功扩增粪产碱杆菌Alcaligenes faecalis NR基因组DNA中amo基因序列,得到长度为1639bp的DNA序列。该段序列包含1个完整的开放阅读框(ORF),编码长度为346aa的多肽链。经过BLAST在线比对分析,该序列为AMO蛋白质序列。该蛋白序列分子量大小为36.7kDa,是碱性蛋白也是疏水性蛋白。此外,AMO也是跨膜蛋白,有12个跨膜区。采用串线法成功构建AMO三级结构模型,模型置信度为98.6%。模型显示AMO含有一个镍原子结合位点。拉氏图表明,AMO模型位于最佳区域的氨基酸残基所占比例为94.9%,这说明模型构象良好。上述研究可为深入了解异养硝化过程中的关键酶提供参考。

Ammonia monooxygenase (AMO) is one of the key enzymes related to nitrification process, and the corresponding gene is amo. In this study, two pairs of primers were designed according to the amo gene sequence in the NCBI database. The amo gene of Alcaligenes faecalis strain NR was amplified successfully, and 1639 bp length DNA sequence was obtained. This sequence contained a complete open reading frame (ORF), which encodes a 346 aa length polypeptide chain. BLAST analysis results showed that this polypeptide was AMO protein sequences. This protein belonged to hydrophobic and basic protein, and the molecular weight was 36.7 kDa. In addition, AMO was transmembrane protein which contained 12 transmembrane regions. Tertiary structure model of AMO was built successfully by using threading method, and the confidence of this model was 98.6%. AMO contained a nickel atom binding sites. The conformation was good according to Ramachandran plot. This investigation offers reference for further understanding the key enzymes relating to heterotrophic nitrification.