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当前位置: 首页 > 文章 > 拟南芥LBD15互作蛋白的筛选和鉴定 华北农学报 2019 (5) 52-58
Position: Home > Articles > 拟南芥LBD15互作蛋白的筛选和鉴定 Acta Agriculturae Boreali-Sinica 2019 (5) 52-58

拟南芥LBD15互作蛋白的筛选和鉴定

作  者：
郭兆来;孙旭东;杨永平;徐慧妮
单  位：
昆明理工大学生命科学与技术学院;中国科学院 昆明植物研究所
关键词：
拟南芥;LBD15;酵母双杂交系统;蛋白互作
摘  要：
为了进一步挖掘拟南芥LBD逆境胁迫响应基因在植物非生物逆境胁迫应答中的作用,探究拟南芥逆境胁迫响应机理,将前期筛选到的LBD15基因构建到pGBKT7酵母表达载体上,筛选与LBD15互作的蛋白.利用酵母双杂交技术筛选了拟南芥全长均一化的酵母文库,在本次筛库试验中,146个样有107个测出序列,通过NCBI Blast分析,其中有96个获得了基因号,占总数的87.9%.根据基因号进行分类,得到了48个蛋白,在筛选到的48个蛋白中有59%的蛋白都是有重复的.然后把基因号通过网站TAIR(www.arabidopsis.org/index.jsp)查找其蛋白序列,通过亚细胞定位预测网站(http://cello.life.nctu.edu.tw/)分析其亚细胞定位,定位分析结果表明,这些蛋白主要定位于叶绿体、细胞质和细胞核.利用Blast2GO进行Gene Ontology注释显示互作蛋白共参与了14个生物过程,包括细胞过程、代谢过程、刺激响应、生物过程的调控、单组织和多组织过程及发育进程调控等.对得到的可能互作蛋白随机挑选了5个进行了互作蛋白的分离和回复验证,结果发现,AP19蛋白及其他一些蛋白的菌落能够在营养缺陷型的平板上正常生长,进一步证实了其为互作蛋白.酵母双杂交文库的成功筛选为进一步研究拟南芥LBD15的分子作用机制奠定了基础.
作  者：
GUO Zhaolai;SUN Xudong;YANG Yongping;XU Huini;College of Life Science and Technology, Kunming University of Science and Technology;Kunming Institute of Botany, Chinese Academy of Sciences;
关键词：
Arabidopsis thaliana;;LBD15;;Yeast two-hybrid system;;Protein interaction
摘  要：
In order to further explore the role of LBD genes in Arabidopsis thaliana in response to plant abiotic stresses, we first inserted LBD15 gene into pGBKT7 vector and constructed the pGBKT7-LBD15 yeast expression vector. The pGBKT7-LBD15 was used to screen the interacted proteins of LBD15. The yeast two-hybrid technique was used to screen a full-length homogenized yeast library of Arabidopsis thaliana. There were 107 detected sequences in 146 samples of which 96 were identified as the Arabidopsis thaliana genes by NCBI Blast, accounting for 87.9% of the total. According to the gene accession number, 48 proteins were obtained, and 59% of them were duplicated. And then the protein sequences were searched via the website TAIR(www.arabidopsis.org/index.jsp) based on the gene accession number. The subcellular localization was analyzed on http://cello.life.nctu.edu.tw/. Through localization analysis, these proteins were mainly located in the chloroplast, cytoplasm and nucleus. Gene Ontology annotations using Blast2 GO showed that the interacting proteins were involved in 14 biological processes, including cellular processes, metabolic processes, stimuli response, regulation of biological processes, single-tissue and multi-tissue processes, and developmental process regulation. Five of the possible interaction proteins were randomly selected and used to verify the interaction. The results showed that the colonies of AP19 protein and other proteins could grow normally on auxotrophic plates, further confirming that they were interacted proteins. The successful screening of yeast two-hybrid library laid the foundation for further study of the molecular mechanism of Arabidopsis LBD15.

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