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

【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1.02×106/3μg pGADT7-RecDNA,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。

[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.