一个棉花锌结合蛋白质(ZnBP)基因的克隆及原核表达

为了解棉花锌结合蛋白的理化性质及结构特征,从已构建完成的棉花(湘棉18)腺体形成时期的cD-NA均一化文库中筛选出1个棉花锌结合蛋白质(Zinc binding protein,ZnBP)基因的全长cDNA序列。经过克隆、测序及序列分析,发现该片段包含1个完整开放阅读框,长654个碱基,编码217个氨基酸,蛋白质分子量为2.46×104,等电点为9.33。以湘棉18叶片DNA为模板,通过PCR扩增分离获得棉花ZnBP基因。经测序拼接,得到1 731 bp的棉花gDNA片段,编码区含有3个内含子。利用pET-28a(+)载体,开展了该基因的原核表达研究,在37℃、异丙基-β-D-硫代吡喃半乳糖苷(IPTG)终浓度1 mmol/L、诱导时间8 h和转速150 r/min条件下,实现最优表达;SDS-PAGE凝胶电泳及Western blotting检测分析结果表明,蛋白表达正确。

Molecular cloning and prokaryotic expression of a zinc-binding protein(ZnBP) in cotton

To study the physical and chemical properties and structural features of zinc-binding proteins(ZnBP) in cotton,the complete sequence of zinc-binding protein was isolated from a normalized cDNA library constructed from cotton(Xiangmian 18) during gland forming stage.The clone was sequenced and analysed.The cDNA fragment is 654 bp encoding 217 amino acid residues,with the molecular weight of 2.46×104 and theoretical pI of 9.33.Cotton ZnBP gene was cloned from gDNA in the leaves of Xiangmian 18.After sequencing,a 1 731-bp cotton ZnBP gene with 3 introns was obtained.Using pET-28a(+) as expression vector,the ZnBP gene prokaryotic expression studies were carried out.The conditions for ZnBP to achieve optimal expression were 37 ℃,1 mmol/L isopropyl β-D-1-thiogalactopyranoside(IPTG),the induction time of 8 hours and the shaker speed of 150 r/min.SDS-PAGE electrophoresis and Western blotting analysis verified the correct expression of the protein.