GFPuv标记猕猴桃溃疡病菌的生物学特性 及其在土壤、根系中的定殖.pdfVIP

中国农业科学 2013,46(2):282-291 Scientia Agricultura Sinica doi: 10.3864/j.issn.0578-1752.2013.02.007 GFPuv 标记猕猴桃溃疡病菌的生物学特性 及其在土壤、根系中的定殖 黄其玲，高小宁，赵志博，秦虎强，黄丽丽 （西北农林科技大学植物保护学院/旱区作物逆境生物学国家重点实验室，陕西杨凌 712100 ） 摘要： 【目的】获得具有荧光标记的猕猴桃溃疡病菌（Pseudomonas syringae pv. actinidiae）菌株，为 进一步揭示其侵染致病过程奠定基础。【方法】电击法对病菌进行GFPuv 基因标记，应用荧光显微镜和平板稀释 法研究标记菌株在土壤和根部的定殖情况。【结果】GFPuv 标记的猕猴桃溃疡病菌菌株在荧光显微镜下发出强烈 的绿色荧光，8 株标记菌基因组DNA 中均扩增出约700 bp 的目的片段。标记菌株PSAmx7-GFPuv1 的菌体形态、生 长曲线、最适温度、最适pH、致病性均与野生型无显著差异，其绿色荧光可稳定遗传。标记菌在灭菌土壤中可存 活3 个月左右，在未灭菌土壤中也能存活3 周；灌根1 d 检测，根表、根内组织中可分离到目标菌落，随后标记 菌株数量呈现“先增后降”的趋势。【结论】电击法成功地将GFPuv 基因转入猕猴桃溃疡病菌；导入的GFPuv 基 因对宿主菌的生物学特性没有影响；标记菌可在灭菌土壤中长期存活，并在根部定殖和增殖。 关键词：猕猴桃溃疡病菌；pDSK-GFPuv；电击转化；土壤存活；根系定殖 Transformed GFPuv into Pseudomonas syringae pv. actinidiae and Its Biological Characteristics and Colonization in Soil and Roots of Kiwifruit HUANG Qi-ling, GAO Xiao-ning, ZHAO Zhi-bo, QIN Hu-qiang, HUANG Li-li (College of Plant Protection, Northwest AF University/State Key Laboratory of Crop Stress Biology for Arid Areas, Yangling 712100, Shaanxi) Abstract: 【Objective 】The objective of this study is to obtain fluorescence-labeled Pseudomonas syringae pv. actinidiae (Psa) strain to further understand the infection and pathogenic processes . 【Method 】The Psa strain was tagged with GFPuv by electroporation. Fluorescence microscope and the method of plate dilution were used to detect the colonization of tagged strain in soil and root. 【Result 】The transformants showed strong green fluorescence under UV-light indicating GFPuv expression. A 700 bp fragment of the GFPuv gene was amplified from the genome DNA of the transformants. There were no diffe