摘要
小麦类甜蛋白基因TaTLP1,在植物抗病防御反应中起重要作用。为筛选与TaTLP1相互作用的蛋白,本研究拟通过Gateway定向克隆技术构建带有GFP标签的重组载体TaTLP1-pEarleyGate103,在烟草中完成异源表达。利用GFP-Trap技术获得与TaTLP1互作的蛋白,质谱分析明确其种类。通过Western blot检测TaTLP1表达情况,以GFP抗体检测,膜上曝光结果显示,在约44 kD处出现条带,证明TaTLP1蛋白在烟草中成功表达;质谱分析GFP-Trap获得的洗脱蛋白,表明获得的候选互作蛋白中定位于胞外的病程相关蛋白1(Pathogenesisrelated protein 1, PR1)、非特异性脂质转移蛋白(Non-specific lipid-transfer protein, nsLTPs)等可能与TaTLP1存在互作。该试验为探索TaTLP1参与小麦抗叶锈病基因Lr19的防御反应机制奠定了理论基础。
Thaumatin-like proteins(TLPs) play an important role in plant defense against pathogenic disease. This study aimed to screen the proteins interacting with TaTLP1.The gene TaTLP1 was ligated into the destination vector pEarleyGate103 with GFP-Tag by Gateway directional cloning method and then proceeded heterologous expression in Nitotiana benthamiana. In order to quickly identify the proteins interacting with TaTLP1, proteins of the TaTLP1-pEarleyGate103 constructs were extracted, and GFP-Trap was used to capture the proteins interacting with TaTLP1. Using GFP antibody to detect the protein, western blot results showed a band around 44 kD indicating the TaTLP1 on the membrane. By mass spectrometry analysis, some extracellular proteins such as PR1 and non-specific lipid-transfer proteins were identified to may interact with TaTLP1. All these results laid the foundation for exploring the defense reaction mechanism of TaTLP1 participating Lr19-mediated resistance to leaf rust.
作者
王菲
杨毅清
武文月
崔钟池
王海燕
刘大群
WANG Fei;YANG Yiqing;WU Wenyue;CUI Zhongchi;WANG Haiyan;Liu Daqun(College of Plant Protection,Hebei Agricultural University/Biological Control Center of Plant Diseases and Plant Pests of Hebei Province,Baoding 071000,China;Graduate School of Chinese Academy of Agricultural Sciences,Beijing 100081,China)
出处
《河北农业大学学报》
CAS
CSCD
北大核心
2020年第2期76-82,共7页
Journal of Hebei Agricultural University
基金
国家自然科学基金(31501623)
河北省高等学校科学技术研究项目(QN2015171).
