桃果实中脱落酸受体ABAR/CHLH结合区蛋白的原核表达、纯化及复性

Prokaryotic expression,purification,and renaturation of abscisi acid-binding protein ABAR/CHLH from peach fruit

为了剖析桃果实中脱落酸受体ABAR/CHLH蛋白的结合活性,从桃果实中提取总RNA,采用RT-PCR方法,以桃果实RNA逆转录的cDNA为模板,扩增出ABAR/CHLH基因的结合区功能片段C369,回收目的片段并测序,基因片段长度为1 121 bp,编码369个氨基酸残基,分子量约为40 kD。利用BamHⅠ和NotI酶切位点将该片段编码区插入原核表达载体pET-28a(+)中,构建ABAR/CHLH基因片段原核表达载体pET28a-C369,经菌落PCR和测序确证后,转化E.coliRosetta(DE3),通过IPTG诱导其表达His-CHLH融合蛋白。通过SDS-PAGE检测及Ni-NTA琼脂糖树脂亲和层析柱纯化目的蛋白,并用纯化复性的His-CHLH C369融合蛋白制备抗体。

To detect the binding activity of ABA receptor ABAR/CHLH in peach,total RNA was extracted from young fruits of Prunus persica.The ABAR/CHLH gene was isolated by RT-PCR using cDNA synthesized from total RNA.The amplified fragment was sequenced to be 1121bp with 369 amino acid residues.This coding sequence was cloned into pET-28a（＋） expression vector.The expression plasmid pET28a-C369 was transformed into E.coli Rosetta（DE3） and expressed in E.coli cells induced by the IPTG.SDS-PAGE analysis revealed that the His-CHLH fusion protein was highly expressed in E.coli Rosetta（DE3）.After purified by Ni-NTA affinity chromatography,the purified protein was successful renaturation,which laid the basis of studying the biological activities related to the protein.