Development and refinement of a high-efficiency gene-targeting system for Aspergillus flavus
Development and refinement of a high-efficiency gene-targeting system for Aspergillus flavus
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DOI：
10.1016/j.mimet.2010.03.010
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发表时间：
2010-06-01
影响因子：
2.2
通讯作者：
Bhatnagar, Deepak
中科院分区：
文献类型：
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作者：
Chang, Perng-Kuang;Scharfenstein, Leslie L.;Bhatnagar, Deepak
建立了一个基于黄曲霉非同源末端连接途径和乳清酸核苷单磷酸脱羧酶基因（pyrG）缺陷的高效基因打靶系统。这是通过用曲霉菌（Aspergillusparathiticus）吡硫胺抗性（ptr）基因替换ku70基因并通过将寄生曲霉（Aspergillusparasiticus）cypA基因插入pyrG基因座来实现的。该系统的效用被证明是通过破坏9个候选基因的分生孢子色素的生物合成。基因靶向频率范围为80%至100%。4号染色体上的两个连锁基因wA和olgA被证实参与色素形成。与产生黄绿色分生孢子的亲本菌株相反，敲除突变体分别产生白色和橄榄绿色分生孢子。通过恢复A中的吡啶硫胺敏感性和尿嘧啶营养缺陷型，进一步完善了该系统。具有工程化pyrG标记的黄曲霉转化受体。这种改进允许使用可重复使用的pyrG标记进行基因操作，如在A. flavus laeA缺失突变体。由爱思唯尔公司出版
An efficient gene-targeting system based on impairment of the nonhomologous end-joining pathway and the orotidine monophosphate decarboxylase gene (pyrG) in Aspergillus flavus was established. It was achieved by replacing the ku70 gene with the Aspergillus oryzae pyrithiamine resistance (ptr) gene and by inserting the Aspergillus parasiticus cypA gene into the pyrG locus. The utility of this system was demonstrated by disruption of nine candidate genes for conidial pigment biosynthesis. The gene-targeting frequencies ranged from 80 to 100%. Two linked genes on chromosome 4, wA and olgA, were confirmed to be involved in pigment formation. In contrast to the parental strain which produced yellowish-green conidia, the knockout mutants produced white and olive-green conidia, respectively. The system was further refined by restoring the pyrithiamine sensitivity and uracil auxotrophy in the A. flavus transformation recipient with an engineered pyrG marker. The improvement allowed gene manipulation using the reusable pyrG marker as shown by the restoration of laeA-mediated aflatoxin production in an A. flavus laeA-deleted mutant. Published by Elsevier B.V.