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小麦F-box/Kelch类基因TaFKOR23的抗逆相关表达模式及分子互作蛋白鉴定
魏春茹1, 孟钰玉1, 范润侨1, 赵梦伊1, 于秀梅1,2,3, 赵伟全3, 康振生2, 刘大群3
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(1.河北农业大学生命科学学院/河北省植物生理与分子病理学 重点实验室,保定 071001;2.西北农林科技大学植物保护学院/旱区作物逆境生物学国家重点实验室,杨凌 712100;3.河北省农作物病虫害生物防治工程技术研究中心,保定 071001)
摘要:
F-box 蛋白在泛素介导的蛋白质降解过程中负责待降解底物的识别,近年来,其在植物抵御逆境中的作用已引起愈 来愈广泛的关注。为了解 F-box 成员在小麦中响应生物和非生物逆境的表达情况及作用机制,本研究从小麦抗叶锈病近等基 因系 TcLr15 中克隆了 F-box 基因 TaFKOR23 的完整编码区序列,该基因编码一个有 421 个氨基酸残基的蛋白,N 端具有 F-box 结构域,C 端带有 2 个明显的 Kelch 结构域,属于 F-box/Kelch 类型基因。系统进化分析表明,TaFKOR23 与粗山羊草(Aegilops tauschii)中的 F-box/Kelch-repeat protein OR23同源性最高,其次与二穗短柄草(Brachypodium distachyon)中的 F-box/Kelch-repeat protein OR23 同源性也较高。利用 qRT-PCR 对接种亲和及非亲和叶锈菌、激素处理、非生物逆境胁迫后 TcLr15 植株中该基因 的表达模式进行分析。研究结果表明,TaFKOR23 基因表达受叶锈菌侵染的诱导,但在亲和与非亲和组合间的表达量无明显差 异;受 ABA、SA 和 MeJA 3 种激素及盐处理后,该基因表达量均呈上调表达趋势,但受 PEG 处理的影响较小;该基因在旗叶 中的表达量远高于其他部位。利用酵母双杂交文库筛选并验证与该基因编码蛋白互作的下游靶蛋白。经文库筛选得到 11 种可 能与 TaFKOR23 互作的靶蛋白,进一步回转验证及β-半乳糖苷酶检测结果表明 TaFKOR23 与 TaSKP1、TaSLY 和 TaChitinase 均存在 相互作用。研究结果为深入解析小麦中 Kelch 类 F-box 基因的功能及代谢网络奠定了基础,并拓宽了对植物中 Kelch 类 F-box 基因的功能认识。
关键词:  小麦  F-box/Kelch  TaFKOR23  表达模式  蛋白互作
DOI:10.13430/j.cnki.jpgr.20190626002
投稿时间:2019-06-26修订日期:2019-12-07
基金项目:旱区作物逆境生物学国家重点实验室开放课题基金(CSBAAKF2018008);河北省高等学校科学技术研究项目(ZD2019086);国家自然科 学基金(31301649);高等学校博士学科点专项科研基金(20121302120010)
Stress-related Expression Profile of F-box/Kelch Gene TaFKOR23 in Wheat and Molecular Characterization of the Interacting Target Protein
WEI Chun-ru1, MENG Yu-yu1, FAN Run-qiao1, ZHAO Meng-yi1, YU Xiu-mei1,2,3, ZHAO Wei-quan3, KANG Zhen-sheng2, LIU Da-qun3
(1.College of Life Sciences, Hebei Agricultural University / Hebei Key Laboratory of Plant Physiology and Molecular Pathology, Baoding 071001;2.College of Plant Protection, Northwest A&F University / State Key Laboratory of Crop Stress Biology for Arid Areas, Yangling 712100;3.Research Center for Engineering and Technology of Biological Control of Pests and Diseases of Agricultural Crops of Hebei Province, Baoding 071001)
Abstract:
F-box protein is responsible for the recognition of substrates to be degraded in the process of ubiquitin-mediated protein degradation. In recent years, the role of F-box protein in plant resistance to stresses has attracted more and more attention. In order to understand the expression profile and molecular mechanism of F-box members in response to biotic/abiotic stresses in wheat, TcLr15 - a near isogenic line against leaf rust pathogen, was used as the material to obtain the complete coding region (encoding a polypeptide of 421 aa) of wheat F-box gene TaFKOR23. Wheat TaFKOR23 includes 1 F-box domain at N-terminal and 2 Kelch domains at C-terminal, which means that the wheat TaFKOR23 belongs to F-box/Kelch subfamily. Phylogenetic tree showed that wheat TaFKOR23 had extremely high homology with F-box/Kelch-repeat protein OR23 from Aegilops tauschii, and had high homology with that from Brachypodium distachyon. Expression profiles of the gene in TcLr15 plants after treatment with leaf rust pathogen, 3 hormones (ABA, SA and MeJA) and abiotic stresses (NaCl and PEG) were analyzed by qRT-PCR in the present study. No significant difference was observed for TaFKOR23 transcripts accumulation between compatible and incompatible combinations; TaFKOR23 showed up-regulated expression after treatment with the 3 hormones and NaCl, but less affected by PEG; TaFKOR23 prominently expressed in flag leaves than in other parts of wheat. Target proteins interacting with TaFKOR23 were screened and identified by yeast two-hybrid, 11 kinds of target proteins were obtained by library screening, and further results showed that TaFKOR23 can interact with TaSKP1, TaSLY and TaChitinase. The research results laid a solid foundation for further understanding the function and metabolic network of F-box/Kelch genes in wheat, and broadened the functional knowledge of Kelch like F-box genes in plant.
Key words:  wheat  F-box/Kelch  TaFKOR23  expression pattern  protein interaction

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