Cloning and Protein Expression of Malate Dehydrogenase Gene of Gossypium hirsuturm L.and Its Enzyme Activity Analysis

doi:10.3969/j.issn.1000-7091.2013.04.003

Abstract

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

CLC Number:

LI Qing, ZHANG Ning，SI Huai-jun， WU Jia-he. Cloning and Protein Expression of Malate Dehydrogenase Gene of Gossypium hirsuturm L.and Its Enzyme Activity Analysis[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2013, 28(4): 13-18. doi: 10.3969/j.issn.1000-7091.2013.04.003.

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