固氮基因nifHDK启动子的功能在大肠杆菌中的验证

Functional Analysis of Promoter of Nitrogen Fixation Gene HDK in Escherichia Coli

近年来,在对肺炎克氏杆菌(Klebsiella pneumoniae)nifA产物研究的基础上已建立了固氮基因调控的模型。固氮基因(nif)的表达受氮调节系统ntr和nifAL操纵子的两个系统的调控。本实验将固氮基因nifHDK启动子、aphA-6基因和rbcL3'构建aphA-6基因表达盒,克隆到pSK.KmR载体,将其转入到大肠杆菌中验证nifHDK启动子的表达活性。结果表明,只有在转入外源nifA基因时,nifHDK启动子才具有转录活性,aphA-6基因才能表达。验证了NifA蛋白和σ54因子是固氮基因的正向调节蛋白,为下一步固氮生物工程研究提供了理论基础。

We have made the model of nitrogen fixation （ nif ） gene regulation on the basis of Klebsiella pneu-moniae nifA protein. The expression of nil gene were regulated by ntr and nifAL. The plasmid pSKKmR was construct by insertion of pnifHDK, aphA-6 and rbcL3＇, then it was transfered into E. coli for testing the expres-sion of pnifHDK. The result illustrated that pnifHDK cant promote gene expression and AphA-6 gene cant ex-press without NifA protein. The result proved that NifA protein and δ54 factor were the up-regulation protein of nif gene. The result also made theoretics foundation of biological nitrogen fixation.