小麦TaSKP2A基因抗逆相关表达分析及与其互作蛋白的筛选  

Anti-stress Related Expression Analysis of TaSKP2A Gene in Wheat(Triticum aestivum) and Its Interaction Protein Screening

作　　者：孟钰玉 李虎滢 许媛 魏春茹 范润侨 于秀梅 赵伟全 康振生[3] 刘大群 MENG Yu-Yu;LI Hu-Ying;XU Yuan;WEI Chun-Ru;FAN Run-Qiao;YU Xiu-Mei;ZHAOWei-Quan;KANG Zhen-Sheng;LIU Da-Qun(College of Life Sciences,Hebei Agricultural University/Key Laboratory of Hebei Province of Plant Physiology and Molecular Pathology,Baoding 071001,China;Biological Control Centre of Plant Disease and Plant Pests of Hebei Province,Baoding 071001,China;College of Plant Protection,Northwest A&F University/State Key Laboratory of Crop Stress Biology for Arid Areas,Yangling 712100,China;Graduate School of Chinese Academy of Agricultural Sciences,Beijing 100081,China)

机构地区：[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]河北省农作物病虫害生物防治工程技术研究中心,保定071001 [3]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 [4]中国农业科学院研究生院,北京100081

出　　处：《农业生物技术学报》2020年第4期571-581,共11页Journal of Agricultural Biotechnology

基　　金：河北省高等学校科学技术研究项目(ZD2019086);旱区作物逆境生物学国家重点实验室开放课题基金(CSBAAKF2018008);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)。

摘　　要：S-期激酶相关蛋白2A (S-phase kinase-associated protein 2A, SKP2A)是一个F-box蛋白,参与植物细胞分裂的调控,并能增加拟南芥(Arabidopsis thaliana)对渗透胁迫的抗性。为探究小麦(Triticum aestivum) SKP2A基因在响应非生物逆境及病原菌侵染中的功能,本研究在克隆到小麦TaSKP2A基因的基础上,对其编码的蛋白进行了分析。结果表明,TaSKP2A编码一条381个氨基酸组成的多肽,预测其等电点和分子量分别为6.83和40.87 kD;TaSKP2A为非分泌型蛋白质,定位于细胞核上,属于富含亮氨酸重复(leucine-rich repeat, LRR)型F-box蛋白。qRT-PCR结果显示,TaSKP2A在雌蕊和旗叶中的表达量较高,在幼茎和雄蕊中较低;经水杨酸(salicylic acid, SA)和NaCl处理后TaSKP2A分别在24和48 h出现上调表达;在H2O2胁迫下,TaSKP2A基因整体呈现下调表达;在叶锈菌(Puccinia triticina)侵染各个时间点的抗病组合中TaSKP2A的表达量高于感病组合,在接种后12 h的抗病组合中,该基因的相对表达量最高,为对照的8.1倍。经酵母(Saccharomyces cerevisiae)文库筛选及回复验证,发现几丁质酶(chitinase, CHI)和SCF (Skp1-Cullins-F-box)复合体骨架蛋白SKP1与TaSPK2A具有相互作用。上述结果提示,TaSKP2A可能响应盐和叶锈菌侵染等逆境胁迫,该F-box蛋白为SCF复合体成员。本研究结果为解析SKP2A蛋白的功能和深入探究其调控网络及作用机制提供了基础资料。S-phase kinase-associated protein 2 A(SKP2 A) is a F-box protein, which is involved in regulating cell division and improving tolerance to osmotic stress in Arabidopsis thaliana. In order to study the role of SKP2 A in the process of wheat(Triticum aestivum) response to abiotic stresses and fungi pathogen infection,the present study further analyzed TaSKP2 A gene and its encoded protein based on the full length ORF of wheat TaSKP2 A. The results showed that TaSKP2 A encoded a polypeptide of 381 amino acids, predicted isoelectric point of TaSKP2 A was 6.83 and the molecular weight was 40.87 kD. TaSKP2 A was non-secreted protein according to the signal peptide prediction, and its subcellular localization was nuclear. TaSKP2 A belonged to F-box/LRR protein based on the C-terminal domain. qRT-PCR results showed that TaSKP2 A was significantly higher expressed in pistil and flag leave, while lower expressed in young stem and stamen of wheat. After treating with salicylic acid(SA) and NaCl, the expression of TaSPK2 A was up-regulated at 24 and48 h, respectively. When treating with H2O2, TaSPK2 A showed gradually down-regulation. The relative expression of TaSPK2 A in resistance combination at all inoculation timepoints were higher than that in susceptible one, and the highest expression(8.1 times higher when comparing to sample at 0 h) occurred in resistance combination at 12 h after inoculation with leaf rust pathogen(Puccinia triticina). Yeast(Saccharomyces cerevisiae) two hybrid was used to screen the target protein interacting with TaSPK2 A, the results showed that chitinase(CHI) and S-phase kinase-associated protein 1(SKP1), a scaffolded protein of Skp1-Cullins-F-box(SCF) complex, could interact with TaSPK2 A. These results suggested that TaSKP2 A might play an important role in response to salt stress and leaf rust pathogen infection, the TaSPK2 A may be a member of SCF complex. The results lay basic foundation for analyzing the functional knowledge of SKP2 A and further exploration of the regulatory network and

关 键 词：小麦 F-BOX蛋白 逆境胁迫 表达模式分析 蛋白互作 

分 类 号：S512.1[农业科学—作物学]