欢迎访问作物学报，今天是
作物学报

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

PDF

1648

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

[1] Aitken A. 14-3-3 proteins: A historic overview. Semin Cancer Biol, 2006, 16: 162-172

[2] Pozuelo-Rubio M, Geraghty K M, Wong B H, Wood N T, Campbell D G, Morrice N, Mackintosh C. 14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking. Biochem J, 2004, 379: 395-408

[3] Schoonheim P J, Veiga H, da Costa Pereira D, Friso G, van Wijk K J, de Boer A H. A comprehensive analysis of the 14-3-3 interactome in barley leaves using a complementary proteomics and two-hybrid approach. Plant Physiol, 2007, 143: 670-683

[4] Alexander R D, Morris P C. A proteomic analysis of 14-3-3 binding proteins from developing barley grains. Proteomics, 2006, 6: 1886-1896

[5] Tetlow I J. Understanding storage starch biosynthesis in plants: a means to quality improvement. Can J Bot, 2006, 84: 1167-1185

[6] Sehnke P C, Chung H J, Wu K, Ferl R J. Regulation of starch accumulation by granule-associated plant 14-3-3 proteins. Proc Natl Acad Sci USA, 2001, 98: 765-770

[7] Zuk M, Weber R, Szopa J. 14-3-3 protein down-regulates key enzyme activities of nitrate and carbohydrate metabolism in potato plants. J Agric Food Chem, 2005, 53: 3454-3460

[8] Tetlow I J, Wait R, Lu Z, Akkasaeng R, Bowsher C G, Esposito S, Kosar-Hashemi B, Morell M K, Emes M J. Protein phosphorylation in amyloplasts regulates starch branching enzyme activity and protein-protein interactions. Plant Cell, 2004, 16: 694-708

[9] Zeng Y, Wu Y, Avigne W T, Koch K E. Differential regulation of sugar-sensitive sucrose synthases by hypoxia and anoxia indicate complementary transcriptional and posttranscriptional responses.Plant Physiol, 1998, 116: 1573-1583

[10] Li Z, Rahman S, Kosar-Hashemi B, Mouille G, Appels R, Morell M K. Cloning and characterization of a gene encoding wheat starch synthase I. Theor Appl Genet, 1999, 98: 1208-1216

[11] Rahman S, Regina A, Li Z, Mukai Y, Yamamoto M, Kosar-Hashemi B, Abrahams S, Morell M K. Comparison of starch-branching enzyme genes reveals evolutionary relationships among isoforms. Characterization of a gene for starch-branching enzyme IIa from wheat D genome donor Aegilops tauschii. Plant Physiol, 2001, 125: 1314-1324

[12] Regina A, Kosar-Hashemi B, Li Z, Pedler A, Mukai Y, Yamamoto M, Gale K, Sharp P J, Morell M K, Rahman S. Starch branching enzyme IIb in wheat is expressed at low levels in the endosperm compared to other cereals and encoded at a non-syntenic locus. Planta, 2005, 222: 899-909

[13] Tetlow I J, Beisel K G, Cameron S, Makhmoudova A, Liu F, Bresolin N S, Wait R, Morell M K, Emes M J. Analysis of protein complexes in wheat amyloplasts reveals functional interactions among starch biosynthetic enzymes. Plant Physiol, 2008, 146: 1878-1891

[14] Toroser D, Athwal G S, Huber S C. Site-specific regulatory interaction between spinach leaf sucrose-phosphate synthase and 14-3-3 proteins. FEBS Lett, 1998, 435: 110-114

[15] Moorhead G, Douglas P, Cotelle V, Harthill J, Morrice N, Meek S, Deiting U, Stitt M, Scarabel M, Aitken A, MacKintosh C. Phosphorylation- dependent interactions between enzymes of plant metabolism and 14-3-3 proteins. Plant J, 1999, 18: 1-12

[16] Esposito D, Chatterjee D K. Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotech, 2006, 17: 353-358

[17] Waugh D S. Making the most of affinity tags. Trends Biotechnol, 2005, 23: 316-320

[18] Pnueli L, Gutfinger T, Haraven D, Ben-Naim O. Tomato SP-interacting proteins define a conserved signaling system that regulates shoot architecture and flowering. Plant Cell, 2001, 13: 2687-2702

[19] Miernyk J A, Thelen J J. Biochemical approaches for discovering protein-protein interactions. Plant J, 2008, 53: 597-609

[20] Hennen-Bierwagen T A, Liu F, Marsh R S, Kim S, Gan Q, Tetlow I J, Emes M J, James M G, Myers A M. Starch biosynthetic enzymes from developing maize endosperm associate in multisubunit complexes. Plant Physiol, 2008, 146: 1892-1908

Kosar-Hashemi B, Li Z, Larroque O, Regina A, Yamamori M, Morell M K, Rahman S. Multiple effects of the starch synthase II mutation in developing wheat endosperm. Funct Plant Biol, 2007, 34: 431-438
[1] 陈松余, 丁一娟, 孙峻溟, 黄登文, 杨楠, 代雨涵, 万华方, 钱伟. 甘蓝型油菜BnCNGC基因家族鉴定及其在核盘菌侵染和PEG处理下的表达特性分析[J]. 作物学报, 2022, 48(6): 1357-1371.
[2] 李海芬, 魏浩, 温世杰, 鲁清, 刘浩, 李少雄, 洪彦彬, 陈小平, 梁炫强. 花生电压依赖性阴离子通道基因(AhVDAC)的克隆及在果针向地性反应中表达分析[J]. 作物学报, 2022, 48(6): 1558-1565.
[3] 李阿立, 冯雅楠, 李萍, 张东升, 宗毓铮, 林文, 郝兴宇. 大豆叶片响应CO2浓度升高、干旱及其交互作用的转录组分析[J]. 作物学报, 2022, 48(5): 1103-1118.
[4] 姚晓华, 王越, 姚有华, 安立昆, 王燕, 吴昆仑. 青稞新基因HvMEL1 AGO的克隆和条纹病胁迫下的表达[J]. 作物学报, 2022, 48(5): 1181-1190.
[5] 周慧文, 丘立杭, 黄杏, 李强, 陈荣发, 范业赓, 罗含敏, 闫海锋, 翁梦苓, 周忠凤, 吴建明. 甘蔗赤霉素氧化酶基因ScGA20ox1的克隆及功能分析[J]. 作物学报, 2022, 48(4): 1017-1026.
[6] 晋敏姗, 曲瑞芳, 李红英, 韩彦卿, 马芳芳, 韩渊怀, 邢国芳. 谷子糖转运蛋白基因SiSTPs的鉴定及其参与谷子抗逆胁迫响应的研究[J]. 作物学报, 2022, 48(4): 825-839.
[7] 袁大双, 邓琬玉, 王珍, 彭茜, 张晓莉, 姚梦楠, 缪文杰, 朱冬鸣, 李加纳, 梁颖. 甘蓝型油菜BnMAPK2基因的克隆及功能分析[J]. 作物学报, 2022, 48(4): 840-850.
[8] 孔垂豹, 庞孜钦, 张才芳, 刘强, 胡朝华, 肖以杰, 袁照年. 不同施肥水平下丛枝菌根真菌对甘蔗生长及养分相关基因共表达网络的影响[J]. 作物学报, 2022, 48(4): 860-872.
[9] 周悦, 赵志华, 张宏宁, 孔佑宾. 大豆紫色酸性磷酸酶基因GmPAP14启动子克隆与功能分析[J]. 作物学报, 2022, 48(3): 590-596.
[10] 黄成, 梁晓梅, 戴成, 文静, 易斌, 涂金星, 沈金雄, 傅廷栋, 马朝芝. 甘蓝型油菜BnAPs基因家族成员全基因组鉴定及分析[J]. 作物学报, 2022, 48(3): 597-607.
[11] 靳容, 蒋薇, 刘明, 赵鹏, 张强强, 李铁鑫, 王丹凤, 范文静, 张爱君, 唐忠厚. 甘薯Dof基因家族挖掘及表达分析[J]. 作物学报, 2022, 48(3): 608-623.
[12] 渠建洲, 冯文豪, 张兴华, 徐淑兔, 薛吉全. 基于全基因组关联分析解析玉米籽粒大小的遗传结构[J]. 作物学报, 2022, 48(2): 304-319.
[13] 陈新宜, 宋宇航, 张孟寒, 李小艳, 李华, 汪月霞, 齐学礼. 干旱对不同品种小麦幼苗的生理生化胁迫以及外源5-氨基乙酰丙酸的缓解作用[J]. 作物学报, 2022, 48(2): 478-487.
[14] 余慧芳, 张卫娜, 康益晨, 范艳玲, 杨昕宇, 石铭福, 张茹艳, 张俊莲, 秦舒浩. 马铃薯CrRLK1Ls基因家族的鉴定及响应晚疫病菌信号的表达分析[J]. 作物学报, 2022, 48(1): 249-258.
[15] 余国武, 青芸, 何珊, 黄玉碧. 玉米SSIIb蛋白多克隆抗体的制备及其应用[J]. 作物学报, 2022, 48(1): 259-264.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2