A simple and effective method for total RNA isolation of appressoria in Magnaporthe oryzae
A simple and effective method for total RNA isolation of appressoria in Magnaporthe oryzae
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一种简单有效的稻瘟病附着菌总RNA分离方法
DOI：
10.1631/jzus.b0860011
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发表时间：
2008-10-01
影响因子：
5.1
通讯作者：
Lin, Fu-cheng
中科院分区：
文献类型：
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作者：
Liu, Tong-bao;Lu, Jian-ping;Lin, Fu-cheng
附着胞的形成是稻瘟病菌与寄主水稻成功互作的重要环节。对附着胞分化过程中发生的分子事件的理解将为水稻稻瘟病的防治提供新的策略。一种快速、可靠的附着胞总RNA提取方法对于研究附着胞形成过程中的基因表达及其机制至关重要。我们发现，重复膜是一个有效的附着胞形成的基质，即使在接种高密度分生孢子。分生孢子接种量为1×106 ml-1时，分生孢子萌发率为（97.98±0.67）%，附着胞形成率为（97.88±0.45）%。在收集附着胞前用Trizol提取总RNA，接种后24 h，从成熟附着胞中提取的总RNA高达113.6 μg。对从cDNA消减文库中分离的两个基因MNH 6和MgATG 1的功能分析表明，RNA的量足以构建cDNA（互补DNA）文库或cDNA消减文库。该方法也适用于其他病原真菌附着胞RNA的提取，这些病原真菌的分生孢子在宿主感染的早期阶段分化为附着胞。
Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1×106 ml−1, the percentages of conidium germination and appressorium formation were (97.98±0.67)% and (97.88±0.45)%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 μg total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA (complementary DNA) library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.