发菜固氮基因nifD和nifK克隆及其失水条件下的差异表达

Cloning of nifD and nifK from Nostoc flagelliforme and theirs Differential Expression under Dehydration

nifD和nifK编码钼铁固氮酶中的钼铁蛋白。为了解发菜nifD和nifK分子信息及对水分胁迫的响应机制,该研究设计了简并性引物克隆发菜nifD和nifK全长,进行原核表达和生物信息学分析,并对不同失水状态下发菜nifD和nifK在转录水平的差异表达和固氮酶活性的变化进行分析。结果表明,发菜nifD和nifK全长分别为1 443 bp和1 536 bp (登陆号为分别为KU886164和KU886165);将nifD和nifK在大肠杆菌中表达,分别获得一个约57 kD和58 kD的外源蛋白;生物信息学分析表明,nifD和nifK核苷酸序列和推译的氨基酸序列均与点形念珠藻(Nostoc punctiforme PCC 73102)高度一致性;nifD和nifK的二级结构主要有α-螺旋、β-折叠、β-转角和随机卷曲。此外,随着藻体含水量的逐渐降低,发菜nifD和nifK在转录水平上的表达量逐渐增加,但固氮酶活性呈现先增加后下降的趋势。研究结果为深入全面研究发菜固氮酶基因结构及其响应水分胁迫的固氮机理及氮代谢途径提供了基础。

Molybdenum-iron protein is coded by nif D and nif K in Mo-Fe nitrogenase. In order to understand molecular information of nifD and nifK and the mechanism in response to dehydration, the full length of nifD and nifK of Nostoc flagelliforme were cloned according to the degenerated primers, as well as prokaryotic expression was carried out and bioinformatics was analyzed. The study also discussed the differential expression at transcription level and changes of nitrogenase activities on dehydration of nifD and nifK of Nostoc flagelliforme under different dehydration situations. The results indicated that, the full length of nifD and nifK were 1 443 bp and 1 536 bp, respectively(GenBank access number is KU886164 and KU886165, respectively). Gene nifD and nifK were expressed in E. coli, and a 57 k D heterologous protein and a 58 kD heterologous protein were obtained, respectively. Bioinformatics analysis suggested that the nucleotide sequence and deduced amino acid sequence of nifD and nifK from Nostoc flagelliforme were similar to that of N. punctiforme PCC 73102 with high consistency. The secondary structure of nifD and nifK were made up of α helix, β-fold, random coil and β-turn. In addition, expression level of nifD and nifK gradually increased at transcription level with the decrease of water content of N. flagelliforme colonies, but nitrogenase activities increased firstly and then decreased. The results laid a foundation for further and comprehensive research on structure of nitrogenase gene and the molecular mechanism of nitrogen fixation as well as nitrogen metabolic pathway for N. flagelliforme in response to dehydration.