拟南芥AFP4的克隆、原核表达和纯化及其与ABI5的互作  

Cloning,prokaryotic expression,purification and interaction with ABI5 of Arabidopsis thaliana AFP4

作　　者：邓子兵[1] 邱梁堃 马建忠[1] DENG Zibing;QIU Liangkun;MA Jianzhong(School of Life Science and Engineering,Lanzhou University of Technology,Lanzhou 730050,China)

机构地区：[1]兰州理工大学生命科学与工程学院,甘肃兰州730050

出　　处：《浙江农业学报》2018年第12期2072-2080,共9页Acta Agriculturae Zhejiangensis

基　　金：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)

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Eco RⅠ和XhoⅠ酶切位点之间和酵母双杂交载体pGBKT7的多克隆位点Eco RⅠ和PstⅠ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Eco RⅠ和PstⅠ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(Gen Bank登录号NM_111081.2)和ABI5基因(Gen Bank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。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 AFP 4 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 ABI 5 gene was inserted into the multiple cloning site of the yeast two-hybrid vector pGADT7.The results of recombinant plasmid showed that the cloned AFP 4 and ABI 5 coding sequences were 100%identical to the AFP 4 gene(GenBank accession number NM_111081.2)and ABI 5 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.

关 键 词：拟南芥 AFP4 原核表达 WESTERNBLOT 酵母双杂交 ABI5 

分 类 号：Q78[生物学—分子生物学]