Lindgren P B, Frederick R, Govindarajan A G, Panopoulos N J, Staskawicz B J, Lindow S E
Department of Plant Pathology, University of California, Berkeley 94720.
EMBO J. 1989 May;8(5):1291-301. doi: 10.1002/j.1460-2075.1989.tb03508.x.
We have constructed derivatives of the transposon Tn3 that allow an ice nucleation gene (inaZ) to be used as 'reporter' of the transcriptional activity of genes into which it is inserted. In these derivatives (Tn3-Ice and Tn3-Spice), the lacZYA sequences of transposon Tn3-HoHo1 were replaced with inaZ lacking its native promoter. The ice nucleation activity of virB::inaZ fusions in the correct transcriptional orientation was inducible by acetosyringone, a plant metabolite which activates the vir operon of Agrobacterium tumefaciens Ti plasmids, while fusions in the opposite orientation were unresponsive to the inducer. Tn3-Spice was also used to investigate the expression of a cluster of genes (hrp) which control pathogenicity and hypersensitivity elicited by Pseudomonas syringae pv. phaseolicola. An inducible region was identified which is expressed at low levels in vitro but becomes activated when the bacteria come into contact with the susceptible host, bean. Activation of this region occurred within 2 h post-inoculation and was nearly complete by the time the bacteria began to multiply in the leaf tissue. The inaZ reporter appears to be at least 10(5)-fold more sensitive than lacZ in P.s.phaseolicola. Thus, the inaZ fusion system provides a sensitive, convenient and inexpensive tool for the study of bacterial gene expression, particularly during plant pathogenesis, and should be generally useful as a reporter gene system in Gram-negative bacteria.
我们构建了转座子Tn3的衍生物，使得冰核基因（inaZ）能够用作其所插入基因转录活性的“报告基因”。在这些衍生物（Tn3-Ice和Tn3-Spice）中，转座子Tn3-HoHo1的lacZYA序列被缺乏其天然启动子的inaZ所取代。正确转录方向的virB::inaZ融合体的冰核活性可被乙酰丁香酮诱导，乙酰丁香酮是一种植物代谢物，可激活根癌农杆菌Ti质粒的vir操纵子，而相反方向的融合体对诱导剂无反应。Tn3-Spice还用于研究控制丁香假单胞菌菜豆致病变种引起的致病性和过敏反应的一组基因（hrp）的表达。鉴定出一个可诱导区域，该区域在体外低水平表达，但当细菌与易感宿主菜豆接触时被激活。该区域的激活在接种后2小时内发生，当细菌开始在叶片组织中繁殖时几乎完成。在菜豆致病变种中，inaZ报告基因似乎比lacZ敏感至少10^5倍。因此，inaZ融合系统为研究细菌基因表达提供了一种灵敏、便捷且廉价的工具，尤其是在植物发病过程中，并且作为革兰氏阴性菌中的报告基因系统应具有普遍用途。
