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【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 nif H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,p I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。
【Objective】 To understand the bioinformatics of Klebsiella variicola DX120E nif H gene from sugarcane endophytic diazotrophs and to provide a theoretical basis for revealing the molecular mechanism of nitrogen fixation of DX120E. 【Method】 Based on the nif H sequence of other nitrogen-fixing strains on NCBI, primers were designed to amplify the endophytic diazotrophs Klebsiella variicola DX120E. The nucleotide sequences and the encoded amino acid sequences were analyzed by bioinformatics method. Its protein structure is predicted. 【Result】 The results showed that the ORF of Klebsiella varicella DX120E nif H gene of sugarcane was 882 bp, Gen Bank accession number was KF732646.1, and the amino acid sequence of nif H gene of six diazotrophs including Klebsiella pneumoniae 342 and Klebsiella variicola At-22 Source up to 95.0% ~ 99.0%. The closest relationship between the DX120E nif H protein and Klebsiella pneumoniae 342 is related to the non-Klebsiella pneumoniae. Bioinformatics analysis showed that the nif H gene of strain DX120E encoded 293 amino acids and the amino acid residues that were particularly important were very conserved. The gene has 11 amino acid residues at the N-terminal, ie tyrosine-glycine-lysine-glycine-glycine-isoleucine-glycine-lysine-serine-threonine-threonine, The ATP motif binding structure. The nif H protein has a molecular weight of 32.07 kD and a pI of 4.92, which is an acidic protein. It has two ASN glycosylation sites, one CAMP phosphorylation site, six CK2 phosphorylation sites and six PKC phosphorylation Site. Hydrophobicity prediction shows that the protein is hydrophilic and has no signal peptide. 【Conclusion】 The deduced amino acid sequence of DX120E nif H gene of Klebsiella variicola has high homology with Klebsiella pneumoniae, which may play an important role in nitrogen fixation of sugarcane effect.