紫云英AD-cDNA文库构建及与豆血红蛋白Lb相互作用靶蛋白的筛选

Construction of Astragalus sinicus AD-cDNA library and identification of proteins interact with the leghemoglobin

摘要:

摘要：【目的】本文旨在构建紫云英酵母双杂交AD-cDNA文库和互作靶蛋白筛选平台，为深入研究共生固氮作用的分子机理奠定工作基础。【方法】以接种华癸中慢生根瘤菌7653R的豆科植物紫云英不同时期根部组织为材料，抽提和纯化RNA，构建了一个酵母AD-cDNA文库。库容量达到1.02×106/3 μg pGADT7-Rec DNA，插入片段大小1-1.5 kb左右。以紫云英豆血红蛋白基因AsB2510构建诱饵载体pGBKT7-AsB2510，利用酵母双杂交技术，筛选与诱饵蛋白相互作用的靶蛋白。【结果】在含有X-gal的SD四缺培养基上筛选得到26个克隆，经过质粒抽提、PCR鉴定、回转酵母验证获得10个阳性克隆。【结论】对阳性克隆的外源片段进行了测序和同源性分析，发现一个值得深入研究的含有tify domain和Divergent CCT motif的转录调控因子。

Abstract:

Abstract: [Objective] The objectives of this work were (i) to construct a yeast two-hybrid AD-cDNA library of Astragalus sinicus and provide a fundamental system to screen target proteins involved symbiotic nitrogen fixation, and (ii) to isolate the target proteins interacting with the leghemoglobin. [Methods] By using the MatchmakerTM Library Construction & Screening Kit (Clontech), we constructed a yeast AD-cDNA library basing on the total RNA, which was isolated from the root and nodule tissues of A. sinicus at different developmental stages infected by Mesorhizobium huakuii 7653R. [Results] The quality examination of the AD-cDNA library showed that the transformation efficiency was 1.0×106 transformants/3μg pGADT7-Rec DNA, and the average length of cDNA inserts was around 1.0 kb. The library was then screened with the leghemoglobin AsB2510 as bait by yeast two-hybrid system, and 26 positive clones was obtained on SD/-Leu/-Trp/-His/-Ade containing X-gal. 10 of them were individually further confirmed by resuing the plasmid, amplifying the cDNA insert and retesting the protein-interacting phenotype. [Conclusions] The cDNA inserts of positive clones were sequenced and undertaken a blast analysis in NCBI database, it was found that clone LY-53 contained a tify domain and divergent CCT motif, which was an important transcription factor needs in-depth investigation.