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致病疫霉菌RXLR效应蛋白AVR1-like寄主靶标的酵母双杂交筛选
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引用本文:张晓将,成 静,单卫星,杜 羽.致病疫霉菌RXLR效应蛋白AVR1-like寄主靶标的酵母双杂交筛选[J].西北农业学报,2021,(1):124~131
DOI:10.7606/j.issn.1004-1389.2021.01.015
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作者单位
张晓将,成 静,单卫星,杜 羽 (1.西北农林科技大学 园艺学院陕西杨凌 7121002.西北农林科技大学 农学院陕西杨凌 712100) 
基金项目:中国博士后项目(2016M600818);国家现代农业产业技术体系项目(CARS-09)。
中文摘要:由卵菌病原物致病疫霉菌引起的马铃薯晚疫病是造成马铃薯毁灭性损失的病害。为了筛选致病疫霉菌RXLR效应蛋白AVR1-like(AL)的寄主靶标,以致病疫霉菌88069菌株的DNA为模板,克隆致病疫霉菌效应蛋白AL,并将其成功构建到pGBKT7载体上,形成诱饵载体。将pGADT7和重组诱饵载体共转化到酵母感受态AH109中,检测到诱饵载体无自激活作用。利用酵母双杂交技术以AL为诱饵筛选晚疫病菌侵染12 h后的番茄cDNA文库,初步获得6个候选AL寄主靶标。对6个候选靶标与AL进行一对一互作验证,结果表明其中有5个与诱饵载体强互作,1个弱互作。这些候选寄主靶标包括热休克蛋白同源蛋白、单脱氢抗坏血酸还原酶蛋白、过氧化物酶体蛋白等。
中文关键词:致病疫霉菌  酵母双杂交  AVR1-like  寄主靶标
 
Yeast Two-hybrid Screening of Host Targets of Phytophthor ainfestans RXLR Effector AVR1-like
Abstract:Late blight disease caused by the Oomycete pathogen Phytophthora infestans is the most devastating disease in potato.In order to screenout the host target of the P.infestans RXLR effector AVR1-like (AL), AL was amplified with the P.infestans strain 88069 DNA as template and the amplified AL sequence was fused to the pGBKT7 vector to form the bait vector.Empty vector pGADT7 and the recombinant bait vector were co-transformed into yeast competent cells AH109 for detecting the self-activation effect,the results showed that the recombinant bait vector didn’t have self-activation activity.The yeast two-hybrid technique was used to screen putative interacting proteins of AL from the tomato cDNA library,mRNAs of tomato leaves infected with P.infestans for 12 hours.Six putative host targets were obtained from the initial screening.The interactions of putative targets with AL were verified using one to one yeast co-transformation, and results showed that five out of the six candidates showed a strong interaction and one candidate showed a weak interaction with AL.These putative host targets included heat shock protein homologous proteins, monodehydroascorbate reductase proteins, peroxisome proteins etc.The identification of six candidate targets of the P.infestans RXLR effector AL provided basis for revealing the virulence mechanism of AL, and for understanding the interaction mechanisms of the oomycete and host plants.
keywords:Phytophthora infestans  Yeast two-hybrid  AVR1-like  Host target
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