| 利用酵母双杂交系统筛选花生AhCaM相互作用蛋白 |
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| 引用本文： | 杨莎,李燕,郭峰,张佳蕾,孟静静,李萌,万书波,李新国.利用酵母双杂交系统筛选花生AhCaM相互作用蛋白[J].作物学报,2015,41(7):1056-1063. |
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| 作者姓名： | 杨莎 李燕 郭峰 张佳蕾 孟静静 李萌 万书波 李新国 |
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| 作者单位： | 1山东省农业科学院生物技术研究中心 / 山东省作物遗传改良与生态生理重点实验室, 山东济南 250100; 2山东省农业科学院人事处, 山东济南 250100 |
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| 基金项目： | 本研究由国家科技支撑计划项目(2014BAD11B04), 山东省农业重大应用技术创新项目, 山东省自主创新成果转化重大专项(2012ZHZXIA0418), 山东省自然科学基金项目(ZR2011CQ042), 山东省农业科学院科技创新重点项目(2014CXZ06-6), 国家现代农业产业技术体系建设专项(CARS-14), 山东省良种产业化工程项目和国家国际科技合作专项(2015DFA31190)资助。 |
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| 摘 要： | CaM是目前已知的胞内Ca2+受体蛋白中最重要的一种,参与多种生理活动的调节。为深入研究CaM蛋白的作用机制,探索其在花生钙信号途径中的作用靶点,用花生CaM基因构建了既无自激活性又无毒性的p GBKT7-AhCaM诱饵表达载体;同时从花生叶片组织中提取总RNA,分离得到mRNA,利用SMART技术合成并纯化双链cDNA后建立花生叶片cDNA文库;通过共转化法筛选与CaM相互作用的蛋白,并对阳性克隆分析和鉴定,筛选到5个与Ah CaM互作的蛋白,其中NAD激酶是已知能与钙调素互作的蛋白;而泛素能与AhCaM互作,说明AhCaM在花生发育及抗逆方面起作用。通过分析这些靶蛋白的已知功能,为研究AhCaM的未知生物学功能提供了重要信息。
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| 关 键 词： | 花生叶片cDNA文库 AhCaM 泛素 酵母双杂交系统 蛋白质相互作用 |
| 收稿时间： | 2014-11-06 |
Screening of AhCaM-Interactive Proteins in Peanuts Using Yeast Two Hybrid System |
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| YANG Sha,LI Yan,GUO Feng,ZHANG Jia-Lei,MENG Jing-Jing,LI Meng,WAN Shu-Bo,LI Xin-Guo.Screening of AhCaM-Interactive Proteins in Peanuts Using Yeast Two Hybrid System[J].Acta Agronomica Sinica,2015,41(7):1056-1063. |
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| Authors: | YANG Sha LI Yan GUO Feng ZHANG Jia-Lei MENG Jing-Jing LI Meng WAN Shu-Bo LI Xin-Guo |
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| Institution: | 1.Biotechnology Research Center of Shandong Academy of Agricultural Sciences / Shandong Provincial key Laboratory of Crop Genetic Improvement, Ecology and Physiology, Ji’nan 250100, China;2.Human Resources Department of Shandong Academy of Agricultural Sciences Ji’nan 250100, China |
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| Abstract: | Calmodulin is the most important known receptor of intracellular Ca2+, which is involved in regulation of many physiological activities. To further study the mechanism of CaM, we isolated and identified proteins that interact with AhCaM by yeast two-hybrid system. The pGBKT7-AhCaM bait vector without toxicity or auto-activation was firstly constructed with the peanut CaM gene. Then, the total RNA of peanut leaf was extracted and the mRNA was isolated, purified to be used as the templates to synthesize ds-cDNA by SMART technology. Ds-cDNA was amplified by long distance PCR. At last, the CaM interaction proteins were screened through co-transformation with bait vector and cDNA prey library. In the peanut cDNA library, five proteins interacting with AhCaM were identified. Among them, NAD kinase was a well-known protein to interact with CaM protein, and ubiquitin could interact with AhCaM, playing a role in development and stressresistance. The significant functional correlation between AhCaM and its interacting proteins peanut will help to elucidate the possible mechanisms of AhCaM in improving the tolerance of transgenic plants. |
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| Keywords: | cDNA library of peanut leaf AhCaM Ubiquitin Yeast two hybrid system Protein interaction |
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