玉米酵母双杂交cDNA文库的构建及ZmCEN互作蛋白的筛选
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国家自然科学基金(21201024);


Construction of Yeast TwoHybrid cDNA Library and Screening of Interaction Proteins of ZmCEN in Maize
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    摘要:

    为阐明玉米中心蛋白(ZmCEN)的生物学功能,采用酵母双杂交技术对其互作蛋白进行研究。提取玉米(Zea mays L.)自交系‘郑58’幼苗的总RNA,利用SMART 技术反转录合成ds cDNA,构建以pGBKT7 为载体的酵母双杂交 cDNA 文库;依据ZmCEN 基因的CDS序列设计引物,构建重组诱饵载体(pGBKT7ZmCEN)转化酵母菌株Y2HGold,检测诱饵载体的毒性与自激活能力后,筛选与玉米中心蛋白(ZmCEN)互作的猎物蛋白。将筛选的互作蛋白NAC67和TONNEAU1b (TON1b)再次验证相互作用,并选取互作蛋白TON1b,采用BiFC实验分别构建ZmCENpSPYNE 和TON1bpSPYCE BiFC半分子重组载体,转化拟南芥原生质体,进一步验证它们在细胞内的互作;并利用 Uniprot 和 KEGG 在线网站对互作蛋白进行 gene ontology(GO)注释分析。结果表明:玉米全株幼苗的cDNA文库库容量达到2.56×107CFU,文库滴度5.36×108 CFU/mL,符合建库要求。经检测诱饵载体无毒性也无自激活功能,所筛选的cDNA 文库经测序和 Blast 比对分析以及共转验证,最终得到28个与诱饵蛋白ZmCEN互作的蛋白质。GO 注释显示互作蛋白参与的生物过程有 21 种。BiFC结果显示,蛋白TON1b与ZmCEN在拟南芥原生质体细胞内互作而形成互补,从而产生黄色荧光,进一步证实了两者存在互作关系。酵母双杂交系统cDNA文库的成功构建与筛选,为进一步研究玉米ZmCEN及其与互作蛋白的作用机制奠定了基础。

    Abstract:

    To understand how Zea mays L. centrin(ZmCEN)function, we employed the technique of yeast two hybrid system (Y2HS) to investigate the interaction between interactingproteins and ZmCEN. In this study, using maize inbred line Zheng 58 seedlings as materials, we isolated the total RNA and synthesized the doublestrand cDNAs by SMART technology. According to the CDS sequence of the ZmCEN gene, we designed the primers to construct recombinant bait vector (pGBKT7ZmCEN) and transform yeast strain Y2HGold for screening the toxicity and selfactivation ability of bait vectors. We screened the prey proteins interaction with ZmCEN. Interacting proteins NAC67 and TON1b were verified the interaction by βgalactosidase assay in vitro. The BiFC semimolecule vectors of ZmCENpSPYNE and TON1bpSPYCE were constructed to further confirm the interaction in Arabidopsis cells. Gene and Ontology (GO) enrichment analysis were performed on the screened proteins using Uniprot and KEGG online sites. The results showed that the unamplified cDNA library was consisted of 2.56 × 107 CFU independent clones, and the titer of library was 5.36 × 108 CFU/mL which met the requirements of library construction. The cDNA library was screened by bait vector after testing no toxicity and no selfactivation, and 28 proteins were interacted with ZmCEN by sequencing and Blast alignment analysis. GO enrichment analysis of these proteins showed that there were 21 terms in the biological process. The ZmCEN and TON1b proteins were further verified interaction in Arabidopsis cell by BiFC technology. A highquality cDNA library was constructed and 28 proteins were interacted with ZmCEN, which could be used to further investigate the interaction mechanisms between them.

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雷海英,白凤麟,段永红,等.玉米酵母双杂交cDNA文库的构建及ZmCEN互作蛋白的筛选[J].西北植物学报,2018,38(4):598-606

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  • 在线发布日期: 2018-05-21
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