Development of a simple and rapid Agrobacterium tumefaciens-mediated transformation system for the entomopathogenic fungus Metarhizium anisopliae var. acridum
Development of a simple and rapid Agrobacterium tumefaciens-mediated transformation system for the entomopathogenic fungus Metarhizium anisopliae var. acridum
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DOI：
10.1111/j.1472-765x.2006.02092.x
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发表时间：
2007-03-01
影响因子：
2.4
通讯作者：
Furlaneto, M. C.
中科院分区：
文献类型：
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作者：
Duarte, R. T. D.;Staats, C. C.;Furlaneto, M. C.
目的：研究农杆菌在共培养条件下对绿僵菌CG423的附着能力，建立农杆菌介导的基因转染方法，将其导入具有生物防治潜力的绿僵菌CG423中。方法与结果：用扫描电镜对农杆菌与金龟子分枝杆菌的共培养进行了分析。我们观察到农杆菌附着在绿僵菌分生孢子和芽管周围并形成聚集体。我们还观察到连接邻近细菌-真菌细胞的纤维样结构的发生。农杆菌介导的转化采用两个携带苯虫酰抗性基因的二元载体作为选择标记。每10(5)个目标分生菌的转化效率可达53个。在非选择性培养基上连续5次转移后，转化子的有丝分裂稳定性很高（89-97%）。分子分析显示该基因发生了高频率的转化。结论：在我们的研究中，我们报道了A. tummefaciens菌株AGL-1附着在昆虫病原真菌绿僵菌（Metarhizium anisopliae var. acridum）上并进行遗传转化。研究的意义和影响：我们首次报道了农杆菌与真菌细胞的附着，为研究T-DNA转移过程的这一重要步骤开辟了新的途径。考虑到本文所描述的转化方案的效率，这是一种有用的工具，用于金龟子分枝杆菌的基因破坏。
Aims: To examine the ability of Agrobacterium to attach to Metarhizium anisopliae var. acridum strain CG423 under co-cultivation and to develop an Agrobacterium-mediated method of gene delivery into strain CG423, a promising agent for biological control of grasshoppers.Methods and Results: The co-cultivation of Agrobacterium tumefaciens and M. anisopliae var. acridum was analysed under scanning electron microscopy. We observed that Agrobacterium attached to and formed aggregates around Metarhizium conidia and germ tubes. We also observed the occurrence of fibril-like structures connecting neighbouring bacterial-fungal cells. The Agrobacterium-mediated transformation was applied using two binary vectors carrying a benomyl resistance gene as a selection marker. The efficiency of transformation was up to 53 transformants per 10(5) target conidia. High mitotic stability of the transformants (89-97%) was demonstrated after five successive transfers on non-selective media. Molecular analysis revealed the occurrence of high frequency of gene conversion.Conclusions: In our study, we report that A. tumefaciens strain AGL-1 attaches to and genetically transforms the entomopathogenic fungus Metarhizium anisopliae var. acridum.Significance and Impact of the Study: We report for the first time, the attachment of Agrobacterium to fungal cells opening new avenues for the study of this essential step of the T-DNA transfer process. Considering the efficiency of the transformation protocol herein described, this is a useful tool for gene disruption in M. anisopliae var. acridum.