摘要： 分析棉花盐胁迫相关的EST文库,设计特异性引物,利用RACE技术从棉花中克隆出苹果酸脱氢酶(Malatedehydrogenase,MDH)基因,命名为GhMDH。基因全长1 130 bp,最大开放阅读框1 014 bp,编码338个氨基酸,分子量为35.495 kDa,等电点pI=8.94。将该基因编码区插入到原核表达载体pET23b中,获得pET23b-GhMDH重组载体。利用IPTG诱导表达目标蛋白,发现通常条件下获得的重组蛋白以包涵体的形式存在。对诱导时间、IPTG浓度和温度等条件进行优化,结果表明,除了在28℃低温以下诱导的蛋白有少量为可溶,其他条件诱导的蛋白均以包涵体形式存在。为了得到足够的重组蛋白,对GhMDH包涵体蛋白采用脲素缓冲液溶解,对提取液进行镍亲和层析柱纯化,对洗脱的产物经脲素梯度透析复性。利用生化测定法测定目标蛋白的酶活性,结果表明,克隆的GhMDH基因编码蛋白酶具有脱氢酶活性。以上结果为GhMDH在棉花体内氧化还原动态以及参与抗逆功能等研究提供前提条件。

关键词: 棉花, 苹果酸脱氢酶, 基因克隆, 诱导表达, 包涵体

Abstract: According to the salt stress- related EST sequences of Gossypium hirsuturm L．，a malate dehydrogen-ase (MDH) gene was isolated by the 3'，5'- RACE technology， named GhMDH．The full- length cDNA of GhMDH is 1 130 bp，containing a 1 014 bp ORF which encodes 338 amino acids．The relative molecular weight of GhMDH protein is 35．495 kDa， and its isoelectric point (pI) is 8．94．The GhMDH ORF had been subcloned into pET23b vector for generating a His- tag fusion recombinant protein． The inducible conditions of recombinant protein expres-sion was optimized，including IPTG contend，temperature and time．The results showed that the target protein was packaged in inclusion body expressed under 37 ℃，1 mmol /L IPTG and 4 hrs induction conditions．The soluble re-combinant protein can only receive in less than 28 ℃ and 12 hrs induction． The urea denatured protein from inclu-sion body had been purified by Ni⁺ column．The purified protein was then renatured through ladder contend dialysis from high to low．The recombinant protein，GhMDH， exhibited high malate dehydrogenase activates via enzymatic as-say．The results is pivotal to investigate GhMDH fuction in cell redox homeostasis and biotic and abiotic stress resist-ance in cotton．

Key words: Gossypium hirsuturm L．, Malate dehydrogenase, Gene clone, Inducide expression, Inclusion body

中图分类号: 

• Q78