甘蔗内生固氮菌Klebsiella variicola DX120E nifH基因的克隆与序列分析

【目的】了解甘蔗内生固氮菌变栖克雷伯氏菌（Klebsiella variicola）DX120E nif H基因的生物信息学,为揭示甘蔗内生固氮菌DX120E的固氮分子机理提供理论依据。【方法】根据NCBI上其他固氮菌株的nif H序列设计引物,对内生固氮菌Klebsiella variicola DX120E菌进行PCR扩增;运用生物信息学方法对其核苷酸序列、编码的氨基酸序列进行分析,并对其蛋白结构进行预测。【结果】甘蔗内生固氮菌Klebsiella variicola DX120E nif H基因的ORF为882bp,Gen Bank登录号为KF732646.1,与Klebsiella pneumoniae 342、Klebsiella variicola At-22等6种固氮菌的nif H基因氨基酸序列同源性高达95.0%-99.0%。DX120E nifH蛋白与Klebsiella pneumoniae 342的亲缘关系最近,与非克雷伯氏菌固氮菌的亲缘关系较远。生物信息学分析显示,菌株DX120E的nif H基因编码293个氨基酸,起特别重要的氨基酸残基非常保守。该基因在N端有11个氨基酸残基即酪氨酸—甘氨酸—赖氨酸—甘氨酸—甘氨酸—异亮氨酸—甘氨酸—赖氨酸—丝氨酸—苏氨酸—苏氨酸,其属于典型结合ATP的基序结构。nif H蛋白分子量为32.07k D,pI为4.92,为酸性蛋白;其具有2个ASN糖基化位点,1个CAMP磷酸化位点,6个CK2磷酸化位点,6个PKC磷酸化位点。疏水性预测显示该蛋白为亲水性蛋白,无信号肽。【结论】变栖克雷伯氏菌（Klebsiella variicola）DX120E nif H基因的推导氨基酸序列与克雷伯氏菌固氮菌属具有较高的同源性,该基因在甘蔗生物固氮过程中可能起重要作用。

Cloning and sequence analysis on nifH gene of Klebsiella variicola DX120E isolated from sugarcane

【Objective 】The present study was aimed to clone nif H gene of Klebsiella variicola DX120 E isolated from sugarcane and conduct bioinformatics analysis of nif H gene in order to provide references for exploiting the ni-trogen- fixing molecular mechanism of endophytic diazotrophs DX120 E in sugarcane. 【 Method 】 According to the nif H 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 DX120 E isolated from sugarcane.The nucleotide sequence, deduced amino acid sequence of nif H gene were analyzed by bioinformatics methods. And its protein structure was also predicted. 【 Result 】 The cloned nif H gene of Klebsiella variicola DX120 E had an ORF of 882 bp, and the gene accession number registered in Gen Bank was KF732646.1. The nif H amino acid sequence of Klebsiella variicola DX120 E 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 nif H gene had higher homology with other nitrogen-fixing species of Klebsiella, but relatively lower with non- Klebsiella species. The bioinformatic analysis results showed that nif H gene of DX120 E encoded 293 amino acids and the important amino acid residues were highly conserved. The nif H 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 nif H protein presented molecular weight of 32.07 k Da, p I 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 nif H protein was hydrophilic protein without signal peptide.【 Conclusion 】 T