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作物学报

作物学报 ›› 2014, Vol. 40 ›› Issue (12): 2192-2197.doi: 10.3724/SP.J.1006.2014.02192

• 耕作栽培·生理生化 • 上一篇    下一篇

籽粒苋AhNAD-ME的序列特征与表达

白云凤1,3,*,聂江婷2,张忠梁1,李平1,张维锋1,闫建俊1,冯瑞云1,张耀1   

  1. 1山西省农业科学院作物科学研究所，山西太原030032；2山西大学生物工程学院，山西太原030006；3农业部黄土高原作物基因资源与种质创制重点实验室，山西太原 030006
  • 收稿日期:2014-02-25 修回日期:2014-09-16 出版日期:2014-12-12 网络出版日期:2014-09-26
  • 通讯作者: 白云凤, E-mail: byfok@126.com
  • 基金资助:

    本研究由国家自然科学基金项目(30971838)，山西省科技攻关项目(201303110015-1)和山西省归国留学人员科研项目(2013-146)资助。

Sequence Characteristics and Expression of NAD-malic enzyme in Amaranthus hypochondriacus L.

BAI Yun-Feng1,3,NIE Jiang-Ting2,ZHANG Zhong-Liang1,LI Ping1,ZHANG Wei-Feng1,YAN Jian-Jun1,FENG Rui-Yun1,ZHANG Yao1   

  1. 1 Institute of Crop Science, Shanxi Academy of Agricultural Sciences, Taiyuan 030032, China; 2 College of Bioengineering, Shanxi University, Taiyuan 030006, China; 3 China Key Laboratory of Loess Plateau Crop Gene Resources and Germplasm Creation, Ministry of Agriculture, Taiyuan 030006, China
  • Received:2014-02-25 Revised:2014-09-16 Published:2014-12-12 Published online:2014-09-26

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摘要：

NAD(P)-苹果酸酶催化苹果酸氧化脱羧，产生丙酮酸和CO2，伴随NAD(P)的还原。在C4植物中，苹果酸酶参与了C4光合作用。本研究对克隆的双子叶C4植物籽粒苋NAD-苹果酸酶基因(AhNAD-ME)编码的氨基酸序列进行了生物信息学分析，结果表明, AhNAD-ME具有苹果酸酶的完整功能域，包括苹果酸N端结构域和苹果酸酶的NAD结合结构域；进化树表明, 该序列属于NAD-ME的α亚基，该亚基定位于线粒体基质中。半定量RT-PCR分析表明，该基因主要在叶片和茎中表达，表达量随光照时间延长而增加。将AhNAD-ME基因重组到原核表达载体pEASY-E1中，电击法转化到大肠杆菌Transette (DE3)菌株中，IPTG诱导其高效表达，表达的融合蛋白的分子量与预期相符，主要以包涵体形式存在。

关键词: 籽粒苋, AhNAD-ME, 序列特征, 表达模式, 原核表达

Abstract:

The NAD(P)-malic enzyme (NAD(P)-ME) found in many metabolic pathways catalyzes the oxidative decarboxylation of L-malate, which results in producing pyruvate, CO2 and NAD(P)H. In C4 plants, NAD(P)-ME plays a key role in photosynthetic carbon fixation. This study was aimed to characterize the AhNAD-ME in dicotyledonous C4 Amaranthus hypochondriacus by sequence analysis, examine the expression patterns of AhNAD-ME gene in different tissues and different durations of illumination time, and construct a recombinant plasmid pEASY-E1 harboring the AhNAD-ME cDNA and then transform the plasmid into E. coli Transette (DE3) for prokaryotic expression after IPTG induction. The result showed that AhNAD-ME contains all of the motifs required for a complete and functional malic enzyme and is localized specifically to the mitochondrial matrix.Semi-quantitative RT-PCR results showed that AhNAD-ME was constitutively expressed in all examined tissues, with different expression levels, and strongly up-regulated under light in the leaf and stem. Results of SDS-PAGE demonstrated that the specific fusion protein with an expected molecular weight was successfully expressed in E. coli transette (DE3) induced by IPTG

Key words: Amaranthus hypochondriacus, AhNAD-ME, Sequence characteristics, Expression pattern, Prokaryotic expression

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