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作物学报 ›› 2009, Vol. 35 ›› Issue (8): 1445-1450.doi: 10.3724/SP.J.1006.2009.01445

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小麦胚乳14-3-3蛋白的表达及其淀粉体淀粉合成酶的互作

宋健民1，戴双1，李豪圣1，刘爱峰1，程敦公1，楚秀生1，Ian J Tetlow2，Michael J Emes2   

  1. 1山东省农业科学院作物研究所，山东济南 250100；2University of Guelph，Guelph，N1G2W1，Canada
  • 收稿日期:2008-12-29 修回日期:2009-03-17 出版日期:2009-08-12 网络出版日期:2009-06-10
  • 基金资助:

    本研究由国家自然科学基金项目（30700492），国家高技术研究发展计划（863计划）项目（2006AA100102），国际科技支撑计划项目（2006BAD01A02），引进国际先进农业科学技术计划（948计划）项目[2006-G2(B)]，公益性行业科研专项（NYHYZX07-002），“泰山学者”建设工程，山东省农业良种工程项目资助。

Expression of a Wheat Endosperm 14-3-3 Protein and Its Interactions with Starch Biosynthetic Enzymes in Amyloplasts

SONG Jian-Min1, DAI Shuang1, LI Hao-Sheng1, LIU Ai-Feng1, CHENG Dun-Gong1, CHU Xiu-Sheng1, Ian J Tetlow2, and Michael J Emes2   

  1. 1Crop Research Institute,Shandong Academy of Agricultural Sciences,Jinan 250100,China;2University of Guelph, Guelph,N2G2W1,Canada
  • Received:2008-12-29 Revised:2009-03-17 Published:2009-08-12 Published online:2009-06-10

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1462

被引次数

7

摘要：

从小麦胚乳中克隆了14-3-3基因，并将其分别插入pET29c和pET41c质粒，用热激法转化大肠杆菌BL21-CodonPlus (DE3)-RP，得到高效表达的蛋白，但融合蛋白主要以包涵体的形式存在。可溶性的融合蛋白可直接通过S-蛋白琼脂糖树脂纯化。包涵体经8 mol L-1尿素溶解变性，稀释复性后，结合到S-蛋白琼脂糖树脂上，也得到纯化的融合蛋白。复性后的融合蛋白对蔗糖合成酶活性表现抑制作用，说明包涵体14-3-3融合蛋白恢复活性。将结合14-3-3融合蛋白的S-蛋白琼脂糖树脂作为诱饵与小麦胚乳淀粉体提取液进行亲和杂交，与14-3-3蛋白特异互作的淀粉合成酶结合到S-蛋白琼脂糖树脂上，Western 检测结果表明, 淀粉体淀粉合酶I(SSI)、淀粉合酶II(SSII)、淀粉分支酶IIa(SBEIIa)、淀粉分支酶IIb(SBEIIb)和ADP焦磷酸化酶大亚基(SH2)与14-3-3蛋白存在互作，而淀粉分支酶I(SBEI)、淀粉磷酸化酶(SP)、D-酶(DE)和ADP焦磷酸化酶小亚基(BT2)不能与14-3-3蛋白结合，说明小麦胚乳14-3-3蛋白对淀粉体淀粉合成具有一定的调控作用。

关键词: 小麦14-3-3蛋白, 表达, 淀粉合成酶, 蛋白互作

Abstract:

Wheat endosperm starch is the major determinant of grain yield and processing quality. The quality and quantity of starch is controlled by a number of starch biosynthetic enzymes. 14-3-3 proteins, involved in many biological processes, are ubiquitous and important regulators in all eukaryotic cells from yeast to mammals and plants. Protein-protein interactions between a wheat endosperm 14-3-3 protein and starch biosynthetic enzymes from amyloplast were investigated in this study. A 14-3-3 gene was cloned from developing wheat endosperm and inserted into plasmid vectors pET29c and pET41c, respectively. The recombinant vectors were transformed into Escherichia coli strain BL21-CodonPlus (DE3)-RP and expressed at very high level. The fusion protein existed mainly as an insoluble inclusion body after extraction by BugBuster Protein Extraction Reagent. The soluble fusion protein was purified by bounding to S-protein agarose, while the inclusion body should be dissolved in 8 mol L-1 urea and refolded firstly. Sucrose synthase activity was shown to be inhibited by exogenous recombinant 14-3-3 protein in a dosage-dependent manner, which suggested the refolded protein was successfully activated and can be used in the following research. The purified recombinant 14-3-3 protein was bound to S-protein agarose as a biochemical bait, and then incubated with wheat amyloplast extract. Proteins interacting specifically with the 14-3-3 protein and remaining on the resin were analyzed by SDS-PAGE and western blotting. These assays showed that starch synthase I (SSI), starch synthase II (SSII), starch branching enzyme IIa (SBEIIa), starch branching enzyme IIb (SBEIIb), and ADP glucose pyrophosphorylase large subunit (SH2) interacted with 14-3-3 protein, whereas SBEI, ADP glucose pyrophosphorylase small subunit (BT2), starch phosphorylase (SP), and D-enzyme (DE) did not bind with the 14-3-3 protein. The results suggest a role for the wheat endosperm 14-3-3 protein in regulation of grain starch biosynthetic enzymes.

Key words: Wheat14-3-3protein, Expression, Starch biosynthetic enzymes, Protein-protein interaction

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