剑麻均一化文库构建及PnRXLR5863靶蛋白筛选  

Construction of Yeast Two-hybrid Normalized Library and Screening of Interaction Proteins of PnRXLR5863 in Agave hybrid No.11648

作　　者：杨子平[1] 杨倩 汪询 柯智 鹿志伟 张燕梅[1] 谌惠邦 周文钊[1] YANG Ziping;YANG Qian;WANG Xun;KE Zhi;LU Zhiwei;ZHANG Yanmei;SHEN Huibang;ZHOU Wenzhao(South Subtropical Crops Institute,Chinese Academy of Tropical Agricultural Sciences/Zhanjiang Key Laboratory of Tropical Crop Genetic Improvement,Zhanjiang,Guangdong 524091,China)

机构地区：[1]中国热带农业科学院南亚热带作物研究所/湛江市热带作物遗传改良重点实验室,广东湛江524091

出　　处：《热带作物学报》2023年第2期246-253,共8页Chinese Journal of Tropical Crops

基　　金：现代农业产业技术体系建设专项资金项目(No.CARS-16);海南省自然科学基金面上项目(No.320MS088);广东省自然科学基金面上项目(No.2022A1515011841)。

摘　　要：剑麻是硬质纤维的主要来源,由烟草疫霉(Phytophthora nicotianae)引起的斑马纹病是剑麻生产的重要病害。RXLR效应蛋白是卵菌第一大类效应蛋白,能通过直接的蛋白质相互作用,靶向寄主防卫相关蛋白,干扰和抑制寄主防卫。利用转录组分析不同侵染阶段的烟草疫霉,发现PnRXLR5863表达量较高,并且其编码的蛋白含有信号肽、RXLR-EER和WY结构域,瞬时表达发现PnRXLR5863能引起烟草细胞死亡,推测PnRXLR5863在烟草疫霉侵染剑麻过程发挥重要作用,但是它作用于剑麻的靶蛋白和致病机制还未知。为了筛选PnRXLR5863的剑麻靶蛋白,应用Switching Mechanism at 5'end of RNA Transcript(SMART)和Duplex-Specific Nuclease(DSN)技术构建了剑麻均一化酵母cDNA表达文库,并筛选了与PnRXLR5863相互作用的蛋白。结果显示,构建的文库库容为5.32×10^(7)CFU/mL,文库总克隆数为1.064×10^(8)CFU,文库片段平均大小为1000 bp,重组率为100%;构建的pGBKT7-PnRXLR5863诱饵载体无自激活性;利用酵母双杂交系统,以pGBKT7-PnRXLR5863诱饵载体,从构建的文库中筛选获得23个与PnRXLR5863相互作用的蛋白。获得的候选靶蛋白主要参与分子功能和生物过程,涉及泛素化过程、蛋白合成、蛋白翻译和蛋白修饰等生理事件,暗示PnRXLR5863效应蛋白可能作用于剑麻的蛋白代谢。剑麻均一化酵母表达文库的构建和PnRXLR5863靶蛋白的筛选,为进一步深入研究PnRXLR5863的致病机理奠定基础。Agave spp.is main resource of hard fiber.Zebra disease,caused by the pathogen Phytophthora nicotianae,is one of the most devastating sisal oomycete diseases.RXLR effector proteins are important cytoplasmic effectors of oomycete and more than 300 RXLR effector genes were identified in the genomes of P.nicotianae.RXLR effectors have the ability to manipulate or suppress host immunity by directly binding host proteins.A typical RXLR effector(named PnRXLR5863)that induced programmed cell death(PCD)was identified from P.nicotianae,but the targets in Agave were still unknown.This study aims to obtain the host plant proteins that are interacting with PnRXLR5863 in A.hybrid No.11648.A normalized cDNA library was constructed by combining Switching Mechanism at 5'end of RNA Transcript(SMART)and Duplex-Specific Nuclease(DSN)technology.The capacity of cDNA library was 5.32×10^(7)CFU/ml,the independent clones was 1.064×10^(8)CFU,and the lengths of inserts ranged from 0.5 kb to 3.0 kb,with the recombination rate of 100%.A total of 23 proteins interacting with PnRXLR5863 were obtained by sequencing and homology analysis.Some of target proteins involve several physiological events,such as ubiquitination,protein synthe-sis,protein translation and protein translation,suggesting that PnRXLR5863 effector may act on protein metabolism of Agave spp.This research would lay a foundation for further study of the pathogenic mechanism of PnRXLR5863.

关 键 词：RXLR效应蛋白 剑麻 均一化文库 蛋白质相互作用 斑马纹病 

分 类 号：S432.1[农业科学—植物病理学]