文档介绍：青枯雷尔氏菌(Ralstonia solanacearum)
绿色荧光蛋白标记研究
车建美1,2,蓝江林1,刘波1
(1福建省农业科学院农业生物资源研究所,福州 350003;2福建农林大学生物农药与化学生物学教育部重点实验室,福州 350002)
摘要:【目的】采用电击法对青枯雷尔氏菌(Ralstoniasolanacearum)进行 gfp/luxAB 基
因双标记,研究标记前后菌株的生长差异、以及侵染性和致病性的变化。【方法】通过电击法进行
青枯雷尔氏菌的遗传转化,采用番茄组培苗对标记菌株的侵染条件和侵染过程进行初步研究。【结
果】成功地采用 gfp/luxAB 基因标记了青枯雷尔氏菌。标记后的菌体细胞形状与亲本菌株形状相同,
均为长椭圆形,近杆状,整个细胞呈现绿色荧光,PCR 结果表明,从标记青枯雷尔氏菌菌株的基因
组 DNA 中扩增出约 kb 的 gfp 基因片段。标记菌在对数生长期,荧光素酶活性快速增加,到稳
定期,荧光素酶活性降低。在无选择压力条件下连续移植 20 次,仍然保持均匀而且强烈的绿色荧
光,荧光稳定性保持在 100%。gfp 基因标记前后的菌株在培养的最适温度、最佳 pH 值及生长曲
线上基本一致,在番茄组培苗内的侵染路线相同,致病力均达到 90%以上。【结论】利用 gfp 和
luxAB 融合基因成功转入青枯雷尔氏菌,融合基因在标记菌株中的表达高效稳定。导入的 gfp 基因
对宿主的生长特性及致病性没有影响。
关键词:青枯雷尔氏菌;电击法;gfp/luxAB 基因
GFP Tagging Ralstonia solanacearum with gfp/luxAB
mini-Tn5
CHE Jian-mei1,2, LAN Jiang-lin1, LIU Bo1
(1Agricultural Bioresource Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350003; 2Key
Laboratory of Biopesticide and Chemical Biology, Fujian Agricultrue and Forestry University, Ministry of Education,
Fuzhou 350002)
Abstract: 【Objective】The Ralstonia solanacearum strain was chromosomally tagged with the marker genes
gfp and luxAB by electroporation. The growth properties and the pathogenicity of the tagged strain and the wild type
strain pared in this paper.【Method】The R. solanacearum strain was transformed by the electroporation. The
condition and processor of the invasion of the GFP-tagged strain was observed in the tomato tissue culture plants.
【Result】 Transformant was screened for the green fluorescence by fluorescence stereomicroscopy. A kb
fragment of the gfp gene and luxAB gene was amplified from the genome DNA of the GFP-tagged strains.
Morphologically, the cells of the engineered strain were similar to wild type strain as determined by laser scanning
confocal microscopy. Luciferase activity of the GFP-tagged strain increased rapidly during the log phase, but
decreased in the stationary phase. The gre