生防放线菌Ahn75的荧光标记及其在水稻中的定殖

【背景】目前gfp标记基因已成为研究靶标微生物与宿主之间互作的一种重要工具。利用gfp基因标记生防菌株,可以对生防菌株的生存及定殖能力进行有效追踪。【目的】对生防放线菌Ahn75进行荧光标记,探讨其在水稻中的定殖规律,为研究Ahn75的稻瘟病防治机制奠定基础。【方法】首先通过电激转化将含绿色荧光标记基因(gfp)的质粒pIJ8655导入大肠杆菌ET12567中,然后采用接合转移的方法将gfp整合到Ahn75基因组上;通过平板对峙试验检验Ahn75-GFP在标记绿色荧光后对稻瘟病病原菌的抑菌活性;采用喷施孢子液的方式将带荧光标记的Ahn75-GFP定殖水稻,并利用荧光显微镜观察生防菌在水稻中的定殖情况;对定殖水稻中的内生菌进行重分离,探究菌株在水稻组织中的分布规律。【结果】PCR扩增和荧光观察表明,绿色荧光标记基因成功整合到生防放线菌Ahn75中。通过平板对峙试验,发现Ahn75-GFP对稻瘟病病原菌抑菌活性与原始菌株没有显著差别。在荧光显微镜下,可以观察到Ahn75-GFP能稳定定殖于水稻的根、茎、叶等组织中,而水稻内生菌重分离试验表明该菌株在茎中的定殖力最强。【结论】获得一株绿色荧光标记生防菌株Ahn75-GFP,结果显示该菌株定殖水稻效果良好,这对于研究Ahn75的稻瘟病防治具有重要意义。

Fluorescent marker of biocontrol actinomycetes Ahn75 and its colonization in rice

Background] At present, marker gene gfp has become an important tool to study the interaction between target microorganism and host. Using gfp to mark the biocontrol strain, we can track the survival and colonization of the biocontrol strain effectively. Objective] The colonization of Ahn75 on rice was studied by making fluorescence labeling in the biocontrol actinomycete Ahn75, to lay a foundation for the resistant mechanism study of Ahn75 against rice blast. Methods] Plasmid pIJ8655 with green fluorescent marker gene (gfp) was introduced into E. coli ET12567 by electroporation, then the gfp gene was incorporated into the genome of Ahn75 through conjugal transfer. The inhibitory activity of Ahn75-GFP against rice blast pathogens was tested by the dural culture method. Ahn75-GFP colonized in rice by spraying spores was observed by fluorescence microscopy, and re-isolated from the rice and counted, to explore the distribution of Ahn75-GFP in rice tissues. Results] The PCR amplification of gfp and the fluorescence observation of Ahn75-GFP showed that, the green fluorescent marker gene was successfully integrated into Ahn75. The inhibitory activity of Ahn75-GFP against rice blast pathogens by the dural culture method was not significantly different from the original strain. Ahn75-GFP colonized in the root, stem and leaf, could be observed by a fluorescent microscope, and the re-isolation results of endophytic strains in rice indicated that the strains had the strongest colonization ability in stems. Conclusion] A green fluorescence-labeled biocontrol strain Ahn75-GFP was successfully obtained, and showed good colonization ability in rice, which was of great significance for studying the control of rice blast by Ahn75 strain.