Improved protocols for functional analysis in the pathogenic fungus Aspergillus flavus.
Improved protocols for functional analysis in the pathogenic fungus Aspergillus flavus.
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改进了病原曲霉曲霉中的功能分析方案。
DOI：
10.1186/1471-2180-7-104
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发表时间：
2007-11-26
期刊：
影响因子：
4.2
通讯作者：
Payne, Gary A.
中科院分区：
文献类型：
--
作者：
He, Zhu-Mei;Price, Michael S.;OBrian, Gregory R.;Georgianna, D. Ryan;Payne, Gary A.
一个可用的全基因组序列的黄曲霉提供了表征因素参与致病性和阐明调控网络参与黄曲霉毒素的生物合成的机会。通过破坏或错误表达靶基因，然后评估其对真菌表型的影响，极大地促进了基因组内基因的功能分析。大规模的功能分析需要一个高效的遗传转化系统和容易选择表达改变的转化体的能力，通常需要产生双（或多）基因缺失菌株或使用原生营养菌株。然而，目前尚缺乏显性选择标记、高效的转化系统和高效的转化体筛选系统。提高了以尿嘧啶营养不良为基础的黄曲霉遗传转化体系的效率。此外，黄芽孢杆菌对抗生素静脉霉素敏感。在100 μg/ml霉素的作用下，用ble基因转化黄曲霉，获得稳定的转化体。通过对尿嘧啶和5-氟糖酸的选择，以及与黄曲霉NRRL 3357中粗神经孢子虫的pyr4基因和A. nidulans的pyrG基因的转化，我们还比较了输尿管霉素系统与基于尿嘧啶互补的输尿管霉素系统。使用pyr4作为可选择标记的转化方案导致靶基因的位点特异性破坏。本研究还开发了一种快速简便的筛选转基因转化子的菌落PCR方法。我们将对霉素的抗性作为黄曲霉遗传转化的一个新的阳性选择标记。本文概述的实验构成了使用抗生素静脉霉素转化黄曲霉的第一份报告。此外，我们证明了这种转化方案可以用于黄曲霉的定向基因破坏。这具有双重意义。首先，它允许菌株在不产生营养缺陷突变的情况下进行转化，这是耗时的，并且可能导致不希望的突变。其次，该方案允许双基因敲除，当使用与现有的菌株营养不良突变。为了进一步促进对该菌株的功能分析，我们开发了一种基于菌落pcr的方法，这是一种快速方便的筛选转基因转化子的方法。这项工作将为黄曲霉毒素代谢的分子生物学研究，特别是基因缺失和基因表达的功能分析提供参考。
An available whole genome sequence for Aspergillus flavus provides the opportunity to characterize factors involved in pathogenicity and to elucidate the regulatory networks involved in aflatoxin biosynthesis. Functional analysis of genes within the genome is greatly facilitated by the ability to disrupt or mis-express target genes and then evaluate their result on the phenotype of the fungus. Large-scale functional analysis requires an efficient genetic transformation system and the ability to readily select transformants with altered expression, and usually requires generation of double (or multi) gene deletion strains or the use of prototrophic strains. However, dominant selectable markers, an efficient transformation system and an efficient screening system for transformants in A. flavus are absent. The efficiency of the genetic transformation system for A. flavus based on uracil auxotrophy was improved. In addition, A. flavus was shown to be sensitive to the antibiotic, phleomycin. Transformation of A. flavus with the ble gene for resistance to phleomycin resulted in stable transformants when selected on 100 μg/ml phleomycin. We also compared the phleomycin system with one based on complementation for uracil auxotrophy which was confirmed by uracil and 5-fluoroorotic acid selection and via transformation with the pyr4 gene from Neurospora crassa and pyrG gene from A. nidulans in A. flavus NRRL 3357. A transformation protocol using pyr4 as a selectable marker resulted in site specific disruption of a target gene. A rapid and convenient colony PCR method for screening genetically altered transformants was also developed in this study. We employed phleomycin resistance as a new positive selectable marker for genetic transformation of A. flavus. The experiments outlined herein constitute the first report of the use of the antibiotic phleomycin for transformation of A. flavus. Further, we demonstrated that this transformation protocol could be used for directed gene disruption in A. flavus. The significance of this is twofold. First, it allows strains to be transformed without having to generate an auxotrophic mutation, which is time consuming and may result in undesirable mutations. Second, this protocol allows for double gene knockouts when used in conjunction with existing strains with auxotrophic mutations. To further facilitate functional analysis in this strain we developed a colony PCR-based method that is a rapid and convenient method for screening genetically altered transformants. This work will be of interest to those working on molecular biology of aflatoxin metabolism in A. flavus, especially for functional analysis using gene deletion and gene expression.
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影响因子：
4.2
作者：
Vogt K;Bhabhra R;Rhodes JC;Askew DS
通讯作者：
Askew DS
影响因子：
4.9
作者：
Cullings, K. W.
通讯作者：
Cullings, K. W.
影响因子：
5.1
作者：
HILLE, J;VERHEGGEN, F;ZABEL, P
通讯作者：
ZABEL, P
影响因子：
4.4
作者：
WOLOSHUK, CP;SEIP, ER;ADKINS, CR
通讯作者：
ADKINS, CR
影响因子：
2.8
作者：
Davidson, RC;Blankenship, JR;Heitman, J
通讯作者：
Heitman, J