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- Title
Cloning and sequence analysis on nifH gene of Klebsiella variicola DX120E isolated from sugarcane.
- Authors
ZHANG Kun-kun; NIU Jun-qi; WEI Chun-yan; XING Yong-xiu; YANG Li-tao; LI Yang-rui
- Abstract
[Objective] The present study was aimed to clone nifH gene of Klebsiella variicola DX120E isolated from sugarcane and conduct bioinformatics analysis of nifH gene in order to provide references for exploiting the nitrogen-fixing molecular mechanism of endophytic diazotrophs DX120E in sugarcane. [Method] According to the nifH gene primers of other nitrogen-fixing bacteria strains registered in the NCBI, the used primer was designed to conduct PCR amplification for endogenous nitrogen-fixing bacterium Klebsiella variicola DX120E isolated from sugarcane. The nucleotide sequence, deduced amino acid sequence of nifH gene were analyzed by bioinformatics methods. And its protein structure was also predicted. [Result] The cloned nifH gene of Klebsiella variicola DX120E had an ORF of 882 bp, and the gene accession number registered in GenBank was KF732646.1. The nifH amino acid sequence of Klebsiella variicola DX120E shared 95.0%-99.0% of homology with the other 6 nitrogen-fixing bacteria viz., K. pneumoniae 342 (YP_002237565.1), D. tsuruhatensis (AAS55953.1), K. oxytoca KCTC 1686(YP_005020938.1), D. dianthicola (WP_024107451.1), Pantoea sp.At -9b (YP_004119245.1), and Dickeya dadantii 3937 (YP_003884784.1). The nifH gene had higher homology with other nitrogen-fixing species of Klebsiella, but relatively lower with non- Klebsiella species. The bioinformatic analysis results showed that nifH gene of DX120E encoded 293 amino acids and the important amino acid residues were highly conserved. The nifH gene had 11 amino acid residues of Tyr-Gly-Lys-Gly-Gly-Ile-Gly-Lys-Ser-Thr-Thr at N terminus, which presented a typical structure of the ATP binding motif. The nifH protein presented molecular weight of 32.07 kDa, pI of 4.92, and belonged to an acidic protein. This protein had 2 ASN glycosylation sites, 1 CAMP phosphorylation site, 6 CK2 phosphorylation sites and 6 PKC phosphorylation sites. The nifH protein was hydrophilic protein without signal peptide. [Conclusion] The deduced amino acid sequence of cloned nifH gene from Klebsiella variicola DX120E shared higher homology with Klebsiella spp. The nifH gene of Klebsiella variicola DX120E maybe play important role in sugarcane biological nitrogen-fixing.
- Subjects
KLEBSIELLA; SUGARCANE; BIOINFORMATICS; POLYMERASE chain reaction; AMINO acids
- Publication
Journal of Southern Agriculture, 2014, Vol 45, Issue 10, p1719
- ISSN
2095-1191
- Publication type
Article
- DOI
10.3969/j:issn.2095-1191.2014.10.1719