| 拟南芥AFP4的克隆、原核表达和纯化及其与ABI5的互作 |
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| 引用本文： | 邓子兵,邱梁堃,马建忠.拟南芥AFP4的克隆、原核表达和纯化及其与ABI5的互作[J].浙江农业学报,2018,30(12):2072. |
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| 作者姓名： | 邓子兵 邱梁堃 马建忠 |
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| 作者单位： | 兰州理工大学 生命科学与工程学院,甘肃 兰州730050 |
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| 基金项目： | 国家自然科学基金(31560073);兰州大学细胞活动与逆境适应教育部重点实验室(lzujbky-2014-bt05) |
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| 摘 要： | 拟南芥ABI5相互作用蛋白AFP4(ABI five binding protein 4)是植物中的一个小分子蛋白,其表达特性与ABI5相似,受ABA诱导。以拟南芥Arabidopsis thaliana L. (Heyn) cv. Columbia为材料,通过PCR扩增了AFP4基因的完整编码序列。扩增片段插入到原核表达载体pET-32a(+)的多克隆位点EcoR Ⅰ和Xho Ⅰ酶切位点之间和酵母双杂交载体pGBKT7的多克隆位点EcoR Ⅰ和Pst Ⅰ酶切位点之间。将ABI5基因的编码序列插入到酵母双杂交载体pGADT7的多克隆位点EcoR Ⅰ和Pst Ⅰ酶切位点之间。重组质粒测序结果表明,所克隆的AFP4和ABI5编码区序列分别与NCBI数据库收录的AFP4基因(GenBank登录号NM_111081.2)和ABI5基因(GenBank登录号NM_129185.3)完全一致。重组原核表达载体含有AFP4片段的重组融合蛋白的理论分子量为53.4 ku。将重组质粒转化至大肠埃希菌表达菌株BL21 Star (DE3)中诱导表达,并经Ni-NTA亲和层析柱分离纯化、SDS-PAGE分析和Western blot鉴定。将含有AFP4和ABI5的酵母双杂交重组质粒共同转化酿酒酵母AH109中进行酵母双杂交。结果表明,重组融合蛋白在大肠埃希菌E. coli BL21 Star (DE3)中表达的适宜条件为:IPTG浓度为0.4 mmol·L-1、25 ℃下诱导表达6 h。在上述条件下,重组融合蛋白占细胞破碎后上清液总蛋白的41.6%。经Ni-NTA亲和层析柱纯化后,AFP4融合蛋白在SDS-PAGE分析时呈现单一条带。该条带经过抗6xHis标签肽的抗体分析,呈阳性。该条带经酵母双杂交分析结果表明,AFP4和ABI5在酵母细胞中可以相互作用。
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| 关 键 词： | 拟南芥 AFP4 原核表达 Western blot 酵母双杂交 ABI5 |
| 收稿时间： | 2018-03-14 |
Cloning,prokaryotic expression,purification and interaction with ABI5 of Arabidopsis thaliana AFP4 |
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| DENG Zibing,QIU Liangkun,MA Jianzhong.Cloning,prokaryotic expression,purification and interaction with ABI5 of Arabidopsis thaliana AFP4[J].Acta Agriculturae Zhejiangensis,2018,30(12):2072. |
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| Authors: | DENG Zibing QIU Liangkun MA Jianzhong |
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| Institution: | School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China |
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| Abstract: | Arabidopsis thaliana ABI5 interacting protein AFP4 (ABI five binding protein 4) is a small molecule protein in plants, and its expression characteristics was similar with ABI5, and was induced by ABA. In this paper, Arabidopsis thaliana L. cv. Columbia was used as a material to amplify the complete coding sequence of AFP4 gene by PCR. The amplified fragment was inserted into the multiple cloning site of prokaryotic expression vector pET-32a(+) and yeast two-hybrid vector pGBKT7. The coding sequence of the ABI5 gene was inserted into the multiple cloning site of the yeast two-hybrid vector pGADT7. The results of recombinant plasmid showed that the cloned AFP4 and ABI5 coding sequences were 100% identical to the AFP4 gene (GenBank accession number NM_111081.2) and ABI5 gene (GenBank accession number NM_129185.3) included in NCBI database. The recombinant molecular weight of recombinant protein containing AFP4 fragment was 53.4 ku. The recombinant plasmids were transformed into Escherichia coli BL21 Star (DE3) and induced, and then analyzed by Ni-NTA affinity chromatography, SDS-PAGE and Western blot. Analysis of yeast two hybridization was carried out as recombinant plasmids containing AFP4 and ABI5 were co-transformed into Saccharomyces cerevisiae AH109. The results suggested that the suitable conditions for the expression of recombinant fusion protein in E. coli BL21 Star (DE3) were as follows: IPTG concentration was 0.4 mmol·L-1,induction at 25 ℃ for 6 h. Under the above conditions, the recombinant fusion protein could account for 41.6% of the E. coli total proteins. After purification by Ni-NTA affinity chromatography column, AFP4 fusion protein showed a single band when analyzed by SDS-PAGE electrophoresis. The band which contained 6×His tag peptide was verified by Western blot. The growth status of colony and the color reaction showed that AFP4 and ABI5 could interact in yeast cells. |
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| Keywords: | Arabidopsis thaliana AFP4 prokaryotic expression Western blot yeast two hybrid ABI5 |
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