Expression of genes involved in germination, conidiogenesis and pathogenesis in Metarhizium anisopliae using quantitative real-time RT-PCR
Expression of genes involved in germination, conidiogenesis and pathogenesis in Metarhizium anisopliae using quantitative real-time RT-PCR
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DOI：
10.1016/j.mycres.2006.04.014
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发表时间：
2006-10-01
影响因子：
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通讯作者：
Bidochka, Michael J.
中科院分区：
文献类型：
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作者：
Fang, Weiguo;Bidochka, Michael J.
绿僵菌（Metarhizium anisopliae）是一种重要的商业昆虫病原真菌，研究其萌发、孢子形成和昆虫发病机制的相关基因是确定提高其功效方法的重要一步。实时RT-PCR是一种灵敏、重复性好、定量研究基因表达的方法。然而，它需要可靠的参考基因转录本进行规范化。本研究选取了act、gpd、18sRNA、tef、try和ubi这6个可能的内参基因作为可靠的内参基因。发现tef、gpd和try转录本是实时RT-PCR分析萌发、分生和发病过程中表达基因的最合适的内参基因。以这些基因为对照，研究了毒力基因、枯草杆菌样蛋白酶（pr1）、参与孢子形成的G蛋白信号调控基因（cag8）、氮反应调控基因（nrr1）和疏水酶基因（ssga）的相对表达水平。这些转录本均未在昆虫发病的早期阶段检测到。氮响应调控因子nrr1在所有发育阶段均一致表达。在昆虫尸体的分生发育后期，cag8的表达水平显著升高。在富营养培养基中，ssga在分生过程中的表达量显著高于菌丝在营养生长过程中的表达量。pr1基因在昆虫尸体真菌分生过程中表达。本研究为利用实时RT-PCR技术研究绿僵菌萌发、分生和发病过程中基因的转录水平奠定了基础。(c) 2006年由Elsevier Ltd代表英国真菌学学会出版。
Characterization of genes involved in germination, conidiogenesis and insect pathogenesis is an important step in identifying methods to increase the efficacy of Metarhizium anisopliae, a commercially important entomopathogenic fungus. Real-time RT-PCR is a sensitive, reproducible and quantitative method to study gene expression. However, it requires reliable reference gene transcripts for normalization. in this study, six putative housekeeping genes (act, gpd, 18sRNA, tef, try and ubi) were investigated as reliable reference genes. Transcripts from tef, gpd and try were found to be the most suitable reference genes for real-time RT-PCR analysis of genes expressed during germination, conidiogenesis and pathogenesis. Using these as reference genes, the relative expression levels of a virulence gene, a subtilisin-like protease (pr1), a regulator of G protein signaling gene involved in conidiogenesis (cag8), the nitrogen response regulator gene (nrr1), and a hydrophobin gene (ssga) were studied. None of these transcripts could be detected in the early stages of insect pathogenesis. The nitrogen response regulator, nrr1, was consistently expressed during all developmental stages. Expression levels of cag8 increased significantly in the later stages of conidiogenesis on insect cadavers. The expression level of ssga during conidiogenesis was significantly higher than that in mycelia during vegetative growth in nutrient rich media. The pr1 gene was expressed during fungal conidiation on the insect cadaver. This study acts as a foundation for investigating the transcriptional levels of genes expressed during germination, conidiogenesis and pathogenesis of M. anisopliae using real-time RT-PCR. (c) 2006 Published by Elsevier Ltd on behalf of The British Mycological Society.