水稻NBS-LRR类抗稻瘟病蛋白Pik-h的互作蛋白筛选

doi:10.3864/j.issn.0578-1752.2016.03.007

摘要/Abstract

摘要： 【目的】利用酵母双杂交系统，以组成抗稻瘟病基因Pik-h介导的抗病反应途径。【方法】以含抗稻瘟病基因Pik-h的近等基因系IRBL8为材料，取稻瘟病菌(Magnaporthe oryzae)GD0193接种12和24 h后的水稻叶片，等量混合后提取总RNA，按照2个紧密连锁且功能独立的Pikh-1和Pikh-2蛋白为诱饵，在水稻叶片中筛选与之互作的蛋白，以便深入研究抗病基因Pik-h的酵母双杂交试剂盒（Make Your Own “Mate&Plate” Library System）的要求构建水稻叶片靶标cDNA文库。利用快速重组克隆的方法构建pGBKT7-Pikh1和pGBKT7-Pikh2诱饵载体，并分别将它们转化至酵母菌株Y2H Gold，提取细胞总蛋白后利用Western blot检测Pikh1和Pikh2的表达情况，并对这2个诱饵载体自激活和毒性分析后进行酵母双杂交筛选。提取SD/-Ade/-Leu/-Trp/ -His/X-α-Gal筛选平板培养基上呈蓝色的酵母单克隆的质粒，将其分别与对应的诱饵载体共转化酵母菌株Y2H Gold，涂布于筛选平板培养基上进行互作的重复验证，将通过重复验证的质粒测序分析所得的序列比对水稻基因组数据库以确定目的基因，并对这些基因进行gene ontology（GO）注释分析以确定其分子功能、生物过程及细胞组成。【结果】靶标cDNA文库的库容量约为2.2×10⁶，插入片段长度均大于400 bp，表明水稻cDNA文库质量高。诱饵载体pGBKT7-Pikh1和pGBKT7-Pikh2均能在酵母细胞中正确表达出对应的Pikh1及Pikh2蛋白,无自激活活性而且对酵母无毒性作用，符合文库筛选的要求。利用含有诱饵载体的酵母菌株Y2H Gold与靶标文库菌株Y187结合（Mating）的方式筛选，获得13个与Pikh-1相互作用的蛋白、5个与Pikh-2相互作用的蛋白，其中有2个与Pikh-1及Pikh-2同时存在相互作用。这些蛋白包括4个在逆境响应或激素信号转导过程中起到重要作用的（辅）转录因子、3个信号蛋白、4个参与光合作用的叶绿体蛋白、1个含有U-BOX结构域的蛋白及4个未知功能蛋白。【结论】成功构建了适宜于研究抗病基因（R基因）介导反应途径的酵母双杂交cDNA文库，筛选出Pik-h的互作蛋白，为进一步研究Pik-h或其他抗稻瘟病基因介导的抗病机制打下了基础。

关键词: 水稻, 稻瘟病菌, 酵母双杂交, 互作蛋白, Pik-h, 筛选

Abstract: 【Objective】 To further investigate the signaling pathways activated directly by the resistance proteins Pikh-1 and Pikh-2 from the resistance gene Pik-h, yeast two-hybrid assay was performed to screen a rice leaf cDNA library using Pikh-1 and Pikh-2 as bait, respectively. 【Method】 All rice leaves of IRBL8 (Pik-h NIL) from the time points (12 and 24 h after inoculation with GD0193) were harvested for total RNA preparation. The rice leaf cDNA library was further constructed with the Make Your Own Mate&Plate Library System kit (Clontech). Bait plasmids pGBKT7-Pikh1 and pGBKT7-Pikh2 were constructed using the rapid homologous recombination method. The expression of the two proteins in the Y2H Gold strain was further detected with Western blot and the self-activation and cytotoxicity were also tested. Proteins interacting with the baits Pikh1 and Pikh2 were screened on SD/-Ade/-His/-Leu/-Trp/X-α-Gal plates from the rice leaf cDNA library with the mating strategy. Then the plasmids were prepared from the blue interacting clones and retransformed back into Y2H Gold strain with the corresponding bait. The plasmids that passed the reconfirmation were sequenced and the DNA sequences were further used to blast rice genome annotation project database. The functions of the interaction proteins were further analyzed with gene ontology (GO) annotation. 【Result】 The rice leaf cDNA library contained 2.2×10⁶ independent clones. The sizes of most inserts were above 400 bp in the cDNA library. Bait plasmids (pGBKT7-Pikh1 and pGBKT7-Pikh2) expressed the corresponding proteins (BD-Pikh-1 and BD-Pikh-2) well and showed no self-activation and cytotoxicity, and suited to this screen. Thirteen candidate interacting proteins of pGBKT7-Pikh1 and five candidate interacting proteins of pGBKT7-Pikh2 were verified. Function annotation showed that four putative proteins were transcription factors involved in stress response and hormone signaling pathways, three were signaling proteins, four were involved in photosynthesis, one U-BOX containing protein was involved in ubiquitin mediated proteolysis, and four were unknown function proteins. 【Conclusion】 A high quality cDNA library was constructed and 16 proteins interacting with Pik-h proteins were identified, and these results provided good clues for elucidating the Pik-h- or other R-gene-mediated disease-resistant mechanisms in rice.

Key words: rice (Oryza sativa), Magnaporthe oryzae, yeast two-hybrid, interacting protein, Pik-h, screening

王加峰，刘浩，王慧，陈志强. 水稻NBS-LRR类抗稻瘟病蛋白Pik-h的互作蛋白筛选[J]. 中国农业科学, 2016, 49(3): 482-490.

WANG Jia-feng, LIU Hao, WANG Hui, CHEN Zhi-qiang . Screening of Putative Proteins That are Interacted with NBS-LRR Protein Pik-h by the Yeast Two-Hybrid System[J]. Scientia Agricultura Sinica, 2016, 49(3): 482-490.

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