利用酵母双杂交系统筛选金针菇DASH型隐花色素互作蛋白  

Yeast Two-Hybrid Screening for DASH-Type Cryptochrome Interaction Proteins in Flammulina filiformis

作　　者：李婧霆 陆欢[2] 李巧珍[2] 徐珍[2] 宋春艳[2] 尚晓冬[2] 王瑞娟[2] 刘建雨 LI Jingting;LU Huan;LI Qiaozhen;XU Zhen;SONG Chunyan;SHANG Xiaodong;WANG Ruijuan;LIU Jianyu(School of Food,Shanghai Ocean University,Shanghai 201400,China;Institute of Edible Fungi,Shanghai Academy of Agricultural Sciences,Key Laboratory of Edible Fungi Resources and Utilization(South),Ministry of Agriculture and Rural Affairs,P.R.China,National Engineering Research Center of Edible Fungi,National R&D Center for Edible Fungi Processing,Shanghai 201403,China)

机构地区：[1]上海海洋大学食品学院,上海201306 [2]上海市农业科学院食用菌研究所,农业农村部南方食用菌资源利用重点实验室,国家食用菌工程技术研究中心,国家食用菌加工技术研发中心,上海201403

出　　处：《食用菌学报》2023年第1期27-35,共9页Acta Edulis Fungi

基　　金：上海市科技兴农项目(G 20200103)。

摘　　要：提取金针菇(Flammulina filiformis)单核体菌株Dan 3菌丝的总RNA,分离纯化mRNA,合成双链cDNA,依次构建酵母双杂交初级和次级cDNA文库。通过酶切连接法构建诱饵载体pGBKT 7-CRY,其无自激活活性和毒性,与构建的次级cDNA文库共转化酵母感受态细胞,筛选与金针菇DASH型隐花色素(FfCRY-DASH)互作的蛋白,对筛选到的6个蛋白进行回补验证,最终发现一个与金针菇DASH型隐花色素互作的蛋白。DASH-type cryptochromes are blue light receptors ubiquitous in animals, plants, and fungi. Using the yeast two-hybrid system, proteins interacting with the DASH-type cryptochrome of Flammulina filiformis(FfCRY-DASH) were screened. The total RNA of F. filiformis monokaryon strain Dan3 was extracted from its mycelia, and then the mRNA was purified to synthesize double-strand cDNA, from which the primary and secondary yeast two-hybrid cDNA libraries were constructed. The recombinant bait vector pGBKT 7-CRY was constructed by restriction endonuclease ligation, and then confirmed that it had no toxicity or autonomous activation activity in yeast Y2HGold cells. Then competent Y2HGold cells were co-transformed with pGBKT7-CRY and the secondary cDNA library to screen prey proteins that interact with FfCRY-DASH. Six candidate interacting proteins were detected and one of them was confirmed by rotary verification. The results provided a reference for the role of FfCRY-DASH in light signal transduction.

关 键 词：金针菇 FfCRY-DASH 酵母双杂交 互作蛋白 

分 类 号：S646[农业科学—蔬菜学]