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小麦条锈菌效应蛋白Pst-1374的表达纯化及初步结构特征
方涵,智东明,赵海斌,王晓杰,康振生,陶虎,邓汉卿,王媛媛
0
(西北农林科技大学生命科学学院,杨凌 712100;西北农林科技大学植物保护学院,杨凌712100;西北农林科技大学化学与药学院,杨凌712100)
摘要:
由条形柄锈菌小麦专化型(Puccinia striiformis f.sp.triticiPst)引起的条锈病是小麦上最具破坏性的病害之一。在条锈菌侵染小麦过程中,条锈菌向小麦细胞分泌效应蛋白,改变宿主的防御反应。效应蛋白Pst-1374来源于条锈菌小种PST-78,将Pst-1374基因插入pET-32a载体并在大肠杆菌中表达,使用镍柱亲和层析和高效液相色谱(HPLC)纯化重组效应蛋白Pst-1374,然后通过液质联用质谱(LC-MS)、动态光散射(DLS)、凝胶过滤层析(GFC)和圆二色谱(CD)研究了Pst-1374的结构特征。结果表明,溶液中的Pst-1374大部分为无规则卷曲结构,只有小部分为规则的二级结构;三氟乙醇(TFE)可以稳定Pst-1374的结构,增加α-螺旋的比例。这些结构特点的发现为其他效应蛋白的结构研究提供了思路。
关键词:  条锈菌  效应蛋白  表达与纯化  本质上非结构蛋白
DOI:10.13610/j.cnki.1672-352x.20200113.007
基金项目:国家自然科学基金(21403168)资助。
Expression, purification and characterization of effector protein PST-1374 from wheat stripe rust
FANG Han,ZHI Dongming,ZHAO Haibin,WANG Xiaojie,KANG Zhensheng,TAO Hu,DENG Hanqing,WANG Yuanyuan
(College of Life Sciences, Northwest A&F University, Yangling 712100;College of Plant Protection, Northwest A&F University, Yangling 712100;College of Chemistry and Pharmacy, Northwest A&F University, Yangling 712100)
Abstract:
Strip rust caused by Puccinia striiformis f.sp.tritici is one of the most devastating diseases in wheat. In the process of infecting wheat with stripe rust, effector proteins were secreted to wheat cells, changing the host's defense response. The effector protein Pst-1374 was derived from Pst isolate PST-78, therefore, Pst-1374 gene was selected to insert into pET-32a vector and expressed in E. coli, and the recombinant effector protein Pst-1374 was purified using Ni2+ affinity chromatography and high performance liquid chromatography (HPLC). The structural characteristics of Pst-1374 were then investigated by liquid chromatography-mass spectrometry (LC-MS), dynamic light scattering (DLS), gel filtration chromatography (GFC) and circular dichroism (CD). The results showed that: most of Pst-1374 in solution is of random coil, and only a small part is in a regular secondary structure; trifluoroethanol (TFE) stabilizes the structure of Pst-1374 and increases the proportion of α-helix. The discovery of these structural features may provide ideas for the study of the structure of other effector proteins.
Key words:  stripe rust  effector protein  expression and purification  intrinsically unstructured proteins

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