棕色固氮菌nifS敲除菌株的构建  

THE CONSTRUCTION AND ANALYSIS OF A nifS DISRUPTION MUTANT

作　　者：汪道涌[1] 谢维[1] 毛晓华[2] 

机构地区：[1]东南大学遗传学研究中心,南京210009 [2]东南大学医学院生物化学与分子生物学系,南京210009

出　　处：《微生物学通报》2003年第6期34-38,共5页Microbiology China

基　　金：东南大学医学基金(No.9223001167)

摘　　要：从Azotobacter vinelandii中通过PCR扩增了5’和3’端分别缺失264bp和261bp的nifS′片段,克隆至载体pUC18,形成重组质粒pUCS,再通过同源重组的方法,将pUCS插入Azoto-bacter vinelandii的nifS中,形成nifS阻断突变体SU1,经Southern杂交和PCR扩增,证明所得确为nifS阻断突变株。SU1在外加氮源的BBGN培养基中能够快速生长,但在Burk's无氮培养基中,生长却极其缓慢,表明nifS基因的破坏,已造成SU1的固氮能力接近完全丧失。该突变体的成功构建,为进一步从中纯化固氮酶两组分,研究nifS对固氮酶结构及功能的影响及iscS与nifS之间的关系奠定了良好的基础。A central portion of nifS, designated nifS', was amplified from Azotobacter vinelandii. The nifS' was cloned into pUC18 to make pUCS. Then pUCS was integrated into Azotobacter vindandii chromosome DNA by homologous recombination. This nifS disruption mutants were generated by single cross-over event and selected by Amp resistence on BBGN medium. The nifS disruption mutant (named SU1) was affirmed by southern blot and PCR amplification. SU1 grows rapidly on BBGN, but very slowly on Burk's N-free medium. This phenomenon showes that SU1 nearly lost its nitrogen fixation ability because of the disruption of nifS. The successful construction of SU1 is helpful for further research on the effect of nifS on the structure and function of nitrogenase component-Ⅰ and Ⅱ.

关 键 词：AZOTOBACTER vinelandii nifS敲除 固氮 

分 类 号：Q936[生物学—微生物学]