关键词: 绿色荧光蛋白, 标记, 青枯劳尔氏菌, 番茄细菌性斑点病菌, 柑橘溃疡病菌

Abstract: 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.

Key words: GFP, labeling, Ralstonia solanacearum, Pseudomonas syringae pv. tomato, Xanthomonas citri subsp. citri