Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens
Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens
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DOI：
10.1139/w06-014
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发表时间：
2006-07-01
影响因子：
2.8
通讯作者：
Bidochka, Michael J.
中科院分区：
文献类型：
--
作者：
Fang, Weiguo;Pei, Yan;Bidochka, Michael J.
建立了一种简便、高效、可靠的农杆菌介导的昆虫病原真菌金龟子绿僵菌遗传转化方法。将绿色荧光蛋白基因egfp和苯菌灵抗性基因benA 3的表达用作转化M.绿僵菌转化效率受菌株的影响。使用根瘤菌。用菌株AGL-1，使用10（6）分生孢子/mL的分生孢子浓度和在200 μ mol/L乙酰丁香酮存在下共培养2天，每个平板可获得17.0 +/-1.4个转化体。另一方面，使用菌株LBA 4404的转化是不成功的。通过Southern分析测试了10个转化体，发现其含有单拷贝T-DNA。将20个转化体在非选择性培养基上继代培养5代，95%的转化体是有丝分裂稳定的。根癌农杆菌介导法转化M.绿僵菌可以作为研究昆虫致病性相关基因的有用工具。
A simple, highly efficient, and reliable Agrobacterium tumefaciens-mediated transformation method was developed for the insect pathogenic fungus Metarhizium anisopliae. Expression of the green fluorescent protein gene, egfp, and the benomyl resistance gene, benA3, were used as markers in transformed M. anisopliae. Transformation efficiencies were dependent on the strain of A. tumefaciens used. With strain AGL-1, 17.0 +/- 1.4 transformants per plate could be obtained using conidial concentrations of 10(6) conidia/mL and a 2 day co-cultivation in the presence of 200 mu mol/L acetosyringone. On the other hand, transformations using strain LBA4404 were unsuccessful. Ten transformants were tested by Southern analysis and found to contain a single copy T-DNA. Twenty transformants were subcultured for five generations on nonselective media, and 95% of the transformants were mitotically stable. Agrobacterium tumefaciens-mediated transformation of M. anisopliae can serve as a useful tool to investigate genes involved in insect pathogenicity.