Differential Expression And Gene Clone Of Nitrogenase Gene (nifH、 NifD And NifK) In N. Flagelliforme Under Drought Stress

Nostoc flagelliforme is kind of terrestrial nitrogen-fixing cyanobacterium, which grows in desert or semi-desert, is a primary resource of local biological nitrogen fixation and pioneer plant. Nitrogenase is encoded by nif, MoFe Nitrogenase is consist of Ferritin and MoFe protein, and Ferritin is encoded by nifH, MoFe protein is encoded by nifD and nifK. The study analyzed the expression changes of nitrogenase gene (nifH、nifD and nifK) in transcriptional level by qRT-PCR for N. flagelliforme under drought stress. Full length of nifH、nifD and nifK were cloned accoding to the degenerated and specific primer, prokaryotic expression was carried out and bioinformatics was analyzed. The results indicated that:(1) The expression level of nifH、nifD and nifK gradually increased in transcriptional level with the decrease of water content in colonies of N. flagelliforme.(2) nifH、nifD and nifK were cloned from N. flagelliforme and a full length of 894 bp,1443 bp, 1536 bp DNA were obtained, respectively (GenBank access number is BankIt 1901364 (KU886163), BankIt1901385 (KU886164), BankIt1901391 (KU886165).respectively).(3) nifH、nifD and nifK were expressed in E. coli, and heterologous protein (36 kD,57 kD and 58 kD) were observed, respectively.(4) Homology analysis showed that each of the nucleotide sequences and amino acid sequences were highly conserved. The alanine of nifH had the strongest hydrophilic in site 240, glutamate had strongest hydrophobicity in site 273. The Lysine of nifD had the strongest hydrophilic in site 28, methionine had strongest hydrophobicity in site 359. The Tyrosine of nifK had the strongest hydrophilic in site 51, Phenylalanine had strongest hydrophobicity in site 76. Ser, Thr and Tys of nifH had four, three, two phosphorylation sites, respectively. Ser, Thr and Tys of nifD had twelve, ten, six phosphorylation sites, respectively. Ser, Thr andTys of nifK had fifteen, eight, six phosphorylation sites, respectively. nifH, nifD and nifK were all extracellular proteins. The secondary structure of nifH, nifD and nifK were made up of a helix, β- fold, β- turn and random coil. Structure prediction of protein tertiary structure showed that the sequence of nifH, nifD and nifK were similar to X diffraction structure, and the similarity were 52%,70% and 55%, respectively. The results laid a foundation for further research on structure of nitrogenase gene and molecular mechanism of nitrogen fixation for N. flagelliforme in response for drought stress.