扩增青枯劳尔氏菌RipAK基因启动子序列,与lacZ基因融合得到pHM1∶PRiPAKLacZ.携带pHM1∶PRiPAKLacZ的青枯劳尔氏菌在营养丰富和基本培养基中都有LacZ活性,表明RipAK启动子可以推动lacZ基因的表达.为构建用于标记植物病原细菌的绿色荧光表达载体,把RipAK启动子和gfp基因克隆到质粒pBBR1MCS-5,使得gfp基因在RipAK启动子的驱动下表达;构建的表达载体pBB-GFP在大肠杆菌中即可表达绿色荧光蛋白.pBB-GFP载体能有效标记青枯劳尔氏菌、番茄细菌性斑点病菌和柑橘溃疡病菌,在荧光显微镜下观察到3种植物病原细菌呈短杆状,青枯劳尔氏菌还可形成多个菌体串联的线状结构.荧光标记对3种病原菌在寄主植物上的致病力没有影响,将标记菌株分别滴加在寄主植物叶片的创伤处,可观察到大量的绿色荧光聚集.本研究构建的pBB-GFP载体能用于多种植物病原细菌的绿色荧光标记,标记后的病原细菌在液体培养及侵染寄主植物过程中都能观察到荧光.%The promoter region of Ralstonia solanacearum RipAK gene was PCR amplified,and fused with lacZ reporter gene to generate pHM1 ∶ PRiPAKLacZ.β-galactosidase (LacZ) activity was successfully detected from R.solanacearum carrying pHM1 ∶ PRiPAKLacZ cultured either in nutrient rich or minimal medium,suggesting that RipAK promoter effectively drove lacZ gene expression.To construct a vector for labeling plant pathogenic bacteria,the RipAK promoter and gfp gene were cloned into plasmid pBBR1MCS-5,which made the expression of gfp gene could be driven by the RipAK promoter.The constructed pBB-GFP was able to express green fluorescent protein GFP in Escherichia coli.Thus,R.solanacearum,Pseudomonas syringae pv.tomato,and Xanthomonas citri subsp.citri were effectively labeled using pBB-GFP.The 3 plant pathogenic bacteria under fluorescence microscopy were short-rod shaped,and occasionally R.solanacearum formed a linear structure in which multiple cells were connected in series.Moreover,GFP labeling had no effect on bacterial pathogenicity on host plants.The labeled strains were inoculated on the wounds of host plant leaves,where a strong green fluorescence was observed at 24 hours post inoculation.In summary,the pBB-GFP vector constructed in this study may be used for labeling several plant pathogenic bacteria with green fluorescence,and the green fluorescence was visualized from labeled bacteria either cultured in medium or inoculated on host plants.
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