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．
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
[1] Goward C R，Nicholls D J．Malate dehydrogenase: A mod-el for structure，evolution and catalysis[J].Prot Sci，1994，3: 1882-1888．
[2] Mauseth J D． Botany，an introduction to plant biology [M]. Florida: Saunders College Publishing，1991: 272-296．
[3] 张 欣． 与植物抗病性有关酶的研究进展[J]. 华南热 带农业大学学报，2000，6(1): 40-46．
[4] Mehdy M C，Sharma Y K． The role of activated oxygen species in plant disease resistance [J]. Physiol Plant，1996，98: 365-374．
[5] Darnell J H，Lodish H． Molecular Cell Biology[M]. New York: Scientific American Books，1986: 904-907．
[6] Wayne A J，John M A，Joseph D G，et al． Stability studies on pig heart mitochondrial malate dehydrogenase: the effect of salts and amino acids [J]. Biochimica ct Bio-physica Acta，1996，1296: 23-34．
[7] 江 滟，王 和． 细胞壁缺陷对伤寒沙门菌苹果酸脱氢酶活性影响的实验观察[J]. 贵州医药，2001，25 (10): 893-894．
[8] Christine G，Michael L． Mitochondrial malate dehydrogen-ase from watermelon: sequence of cDNA clones and pri-mary structure of the higher-plant precursor protein[J]，Plant Molecular Biology，1990，14: 1019-1030．
[9] 姜瑞华． 水稻苹果酸脱氢酶基因的克隆及其在 E． coli 中的表达[J]. 复旦学报，2002，41(1): 67-69．
[10] Haas B J，Volfovsky N，Town C D，et al． Full-length mes-senger RNA sequences greatly improve genome annota-tion[J]. Genome Biol，2002，3(6): 0029． 1-0029． 12．
[11] 张 觅． 香橙苹果酸脱氢酶 CjMDH 基因表达特性及 转基因烟草的耐铝性分析[J]. 园艺学报，2008，35 (12): 1751-1758．
[12] 贾 晋． 大白菜苹果酸脱氢酶基因的克隆及序列分 析[J]. 园艺学报，2009，36(3): 363-368．
[13] 邓秋菊． 香蕉果实采后成熟过程中苹果酸脱氢酶及苹果酸含量的变化[J]. 热带农业科学，2011，31(7): 34-38．
[14] 陈露露． 茶树胞质性苹果酸脱氢酶的原核表达及生 物信息学分析[J]. 云南植物研究，2010，32 (1): 32-40．
[15] 李 晶，王亦学，郑德刚，等． 一种简单高效提取棉花 不同组织总 RNA 的方法[J].山西农业科学，2009，37(5): 17－19．
[16] Shan L W，Tang R C，Liu S Y，et al．Prokaryotic expres-sion，purification and preparation of polyclonal antibody for wheat grain peroxidase WP1 gene[J].Chin J Bio-tech，2011，27(2): 26－30．
[17] Chang B M，Liu X J，Li M N，et al．Expression，purifica-tion，renaturation and activity assay of NK4 in Escherich-ia coli[J].Fudan Univ J Med Sci，2011，38(1): 60-65．
[18] Yao X Y，Li M，Zhai H，et al．Isolation and characteriza-tion of an apple cytosolic malate dehydrogenase gene re-veal its function in malatesynthesis[J].Journal of Plant Physiology，2010，168(5): 474－480．
[19] 王晓云，毕玉芬． 植物苹果酸脱氢酶研究进展[J]. 生 物技术通报，2006，4: 44－47．
[20] 贺庆华，郑玉才．盐生杜氏藻D．salina 和D．bardwil 苹 果酸脱氢酶基因的克隆和序列分析[J].安徽农业科 学，2008，36(11): 4435－4436．
[21] William S，Song T．Recombinant Protein Complex Ex-pression in E．coli[J].Current Protocols in Protein Sci-ence，2008，21(5): 324－321．
[22] Weikai L T，Jorge E C．Activation of partially folded mi-tochondrial malate dehydrogenase by thioredoxin[J]. Eur J Biochem，1997，246: 127－132．
23] Cheung P Y，Churchich J E．Recognition of Protein Sub-strates by Protein-disulfide Isomerase[J].Journal of Bi-ological Chemistry，1999，274(46): 32757-32761.
[24] 张志远． 猪 Fc 受体 亚单位基因克隆与原核表达 [J]. 华北农学报，2012，27(1): 35-38．
[25] Wiley J，ons I．Preparation and Extraction of Insoluble (Inclusion Body) Proteins from Escherichia coli [J]. Current Protocols in Protein Science，2004，16(4): 131-143．
[26] Tesfaye M，Temple S J，Allan D L，et al．Overexpression of malate dehydrogenase in transgenic alfalfa enhances organic acid synthesis and confers tolerance to aluminum [J].Plant Physiol，2001，127(4): 1836－1844．
