番茄抗叶霉病基因Cf-12的亚细胞定位和互作蛋白的筛选  

Subcellular localization of tomato leaf mould resistance gene Cf-12 and screening of interacting proteins

作　　者：张新宇 刘晗 谢欣欣 孙梦琳 蒋嘉宁 杜南山 薛东齐 ZHANG Xinyu;LIU Han;XIE Xinxin;SUN Menglin;JIANG Jianing;DU Nanshan;XUE Dongqi(College of Horticulture,Henan Agricultural University,Zhengzhou 450002,China;College of Tropical Agriculture and Forestry,Hainan University,Haikou 571737,China)

机构地区：[1]河南农业大学园艺学院,郑州450002 [2]海南大学热带农林学院,海口571737

出　　处：《东北农业大学学报》2025年第1期42-49,共8页Journal of Northeast Agricultural University

基　　金：国家自然科学基金项目(32472728,32202486);河南省重点研发与推广专项(232102110011)。

摘　　要：叶霉菌引起的叶霉病是番茄的重要病害之一,现有的Cf抗病基因易被新分化的叶霉菌小种克服。接种鉴定发现Cf-12高抗多个叶霉菌生理小种,但其抗病机制有待解析。研究首先对Cf-12基因进行亚细胞定位,利用酵母双杂交膜系统筛选Cf-12互作蛋白。结果表明:Cf-12蛋白定位在细胞膜上,虽成功构建诱饵质粒p BT3-SUC-Cf12,但该诱饵未通过蛋白功能验证,无法进行酵母膜体系筛库试验;遂构建p GBKT7-Cf12诱饵质粒进行酵母双杂交核文库筛选,诱饵质粒对Y2H Gold酵母无毒且无自激活活性。将含p GBKT7-Cf12的诱饵质粒酵母菌与番茄pGADT7-cDNA文库质粒进行共转筛库,初筛获得16个克隆,复筛获得4个阳性克隆,编码基因分别为Solyc06g062370.3、Solyc12g005850.2、Solyc06g071370.1和Solyc03g093690.3。Tomato leaf mould caused by Cladosporium fulvum is a major disease affecting tomatoes,and the Cf resistance genes of commercial varieties are often overcome by newly differentiated C.fulvum races.Inoculation identification showed that Cf-12 was highly resistant to multiple C.fulvum races,but its disease resistance mechanism remains not clear.In this study,the subcellular localization of Cf-12 gene was performed,and the interacting protein of Cf-12 was screened using the yeast two-hybrid membrane system.The results showed that Cf-12 proten was localized on the cell membrane,and the bait plasmid pBT3-SUC-Cf12 was successfully constructed.The functional verification failed of bait protein,so the membrane system screening experiment could not be performed.Therefore,pGBKT7-Cf12 for yeast two-hybrid nuclear library screening was constructed.The Cf-12 protein was non-toxic to Y2H Gold yeast and exhibited no self-activation activity.Through the screening of tomato nuclear cDNA libraries,a total of 16 clones were obtained from primary screening and four positive clones were obtained by re-screening,and the coding genes of the four positive clones were Solyc06g062370.3,Solyc12g005850.2,Solyc06g071370.1 and Solyc03g093690.3,respectively.

关 键 词：番茄 叶霉病 Cf-12 酵母双杂交 互作蛋白 

分 类 号：S641.2[农业科学—蔬菜学]