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Functional genomics in the rice blastfungus, Magnapathe oryzae using an RNA siencing approach

使用 RNA 测序方法对稻瘟菌 Magnapathe oryzae 进行功能基因组学研究

基本信息

批准号：
17380030
负责人：
NAKAYASHIKI Hitoshi
金额：
$ 10.8万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17380030/
关键词：
RNAi RNA silencing Maonaoorthe orvzae ポストゲノム病原性遺伝子

项目摘要

We developed an RNA silencing vector, pSilent-Duall (pSD1), with a convergent dual Pol II promoter system that provides a high-throughput platform for functional genomics research in filamentous fungi. In the pSD1 system, the target gene was designed to be transcribed as a chimeric RNA with enhanced green fluorescent protein (eGFP) RNA. This enabled us to efficiently screen the resulting transformants using GFP fluorescence as an indicator of gene silencing. A model study with the eGFP gene showed that pSD1-based vectors induced gene silencing via the RNAi pathway with slightly lower efficiency than did hairpin eGFP RNA-expressing vectors. To demonstrate the applicability of the pSD1 system for elucidating gene function in the rice blast fungus Magnaporthe oryzae, 37 calcium signaling-related genes that include almost all known calcium signaling proteins in the genome were targeted for gene silencing by the vector. Phenotypic analyses of the silenced transformants showed that at least 26, 35, and 15 of the 37 genes examined were involved in hyphal growth, sporulation and pathogenicity, respectively, in M oryzae. These included several novel findings such as that Pmcl-, Spfl- and Neo1-like Ca^<+>2 pumps, calreticulin, and calpactin heavy chain were essential for fungal pathogenicity.

我们开发了一种RNA沉默载体pSilent-Duall (pSD1)，它具有聚合双Pol II启动子系统，为丝状真菌的功能基因组学研究提供了一个高通量平台。在pSD1系统中，目的基因被设计成与增强的绿色荧光蛋白（eGFP） RNA嵌合转录。这使我们能够使用GFP荧光作为基因沉默的指标有效地筛选产生的转化子。一项eGFP基因的模型研究表明，psd1载体通过RNAi途径诱导基因沉默，其效率略低于发夹eGFP rna表达载体。为了证明pSD1系统在水稻稻瘟病菌中阐明基因功能的适用性，该载体将37个钙信号相关基因（包括基因组中几乎所有已知的钙信号蛋白）作为基因沉默的目标。对沉默转化体的表型分析表明，37个基因中至少有26个、35个和15个分别与米孢霉菌丝生长、孢子形成和致病性有关。其中包括一些新发现，如Pmcl-， Spfl-和neo1样Ca^<+>2泵，钙网蛋白和钙凝蛋白重链对真菌致病性至关重要。