CLONING,PROKARYOTIC EXPRESSION AND PROTEIN PURIFICATION OF SUGARCANE ENDOGENOUS AZOTOBACTER KLEBSIEL

• 作 者: Kun-Kun Zhang Yong-Xiu Xing Min Shao Chao-Xing Wu Li-Tao Yang Yang-Rui Li
• 机 构: State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources,College of Ag
• 出 处: 广西甘蔗学会2016年年会暨学术交流会
• 发表日期: 2016年1期
• 关键词: Sugarcane Klebsiella variicola DX120E clone of nifK gene prokaryotic expression

　　NifK gene is a key nitrogenase and plays an important role in the nitrogen fixation process.It is necessary to obtain the nifK gene for exploring its relationship with nitrogen fixation.This study aimed to clone nifK gene,predict the secondary structure,the characters and functions of the nifK protein,and conduct prokaryotic expression.According to other nifK sequences registered in the NCBI,specific primers were designed.Klebsiella variicola DX120E nifK gene opening reading frame(ORF)was cloned by PCR amplification,and its nucleotide sequence,amino acid sequence and protein structure were analyzed by combining bioinformatics methods.Prokaryotic expression vector pET30a(+)was used to construct the recombinant expression vector pET30a-nifK.After PCR,double enzyme digestion and DNA sequencing,the recombinant plasmid was transformed into BL21(DE3),and 1.0 mmol/L IPTG was used to induce the gene expression at 28 ℃.The fusion protein expression was detected through SDS-PAGE electrophoresis.The nifK gene ORF cloned from Klebsiella variicola DX120E in this study was 1563 bp,encoding 520 amino acids.The prokaryotic expression protein by induction was detected by mass spectrometry,which had an isoelectric point of 5.54,and molecular weight of 58.12 kDa.The gene has been registered in GenBank with accession number KF732647.Amino acid sequence comparison and phylogenetic tree analysis showed that the nifK gene had the highest similarity with Klebsiella.The nifK gene was cloned and the nifK protein was successfully expressed in a prokaryotic expression system,which laid the foundation for further studying the function of DX120E nifK gene in sugarcane.