列当生防菌Br-2的DsRed红色荧光标记及定殖

Broomrape bio-control agent Br-2 labeled by red fluorescent protein and its colonization

为探明生防菌Br-2在分枝列当体内及番茄根围的定殖能力,采用PEG-CaCl_2介导的原生质体转化体系将红色荧光蛋白基因DsRed转入到菌株Br-2中,并构建其生长曲线,测定其对分枝列当种子萌发的抑制率,通过共聚焦显微镜观察其在分枝列当茎内及番茄根围的定殖情况。结果表明:通过原生质体转化体系得到了标记菌株Br-2-DsRed,继代培养5代后,其在共聚焦显微镜下仍可观察到亮红色荧光,经PCR检测带有DsRed基因,证明其稳定性好。标记菌株Br-2-DsRed与野生菌株Br-2的生长曲线基本一致,对分枝列当种子萌发的抑制率分别为75.86%和74.14%,无显著差异。显微观察发现,标记菌株Br-2-DsRed接种1 d后即能够侵染分枝列当的茎部表皮,3 d后主要分布在茎表皮和厚壁细胞间隙,5 d后侵染到厚壁细胞及维管束细胞内部并导致茎部腐烂;而标记菌株Br-2-DsRed不能侵染番茄根尖的内部,主要定殖在番茄根尖表面。

To investigate the colonization of the broomrape bio-control agent Fusarium oxysporium （Br-2） in the Orobanche aegyptiaca stem and tomato root tip, the gene of red fluorescent protein （RFP/ DsRed） was transferred in the strain Br-2 by the PEG-CaC12-mediated protoplast transformation system. Dynamic growth analyses and inhibiting effect on germination to O. aegyptiaca seed were tested in this study. Br-2-DsRed colonization in the O. aegyptiaca stem and tomato root tip was conducted by the la- ser scanning eonfocal microscope. The results showed that the labeled strain Br-2-DsRed was obtained by the protoplast transformation system. After five generations subcutturing, bright red fluorescence could still be observed under the confocal microscope and DsRed genes could be detected by PCR in the strain Br-2-DsRed. And the growth curve of labeled strain Br-2-DsRed was basically same as the wild stain Br-2, there was no significant difference in the inhibiting effect on germination to O. aegypti- aca seed between labeled strain Br-2-DsRed and wild strain Br-2 with the inhibition rates were 75.86% and 74.14%, respectively. By the laser scanning confocal microscope, Br-2-DsRed strain infected the epidermis of O. aegyptiaca stem one day after inoculation, infected the intercellular of the epidermis and sclerenchymatous cell three days after inoculation, finally Br-2-DsRed strain infected the cell of the sclerenchymatous and vascular bundle and lead stem to rot after five days. But it mainly distributed on the surface of tomato root couldn＇t infect the interior of the root tip.