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

1923

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] 崔致远, 秦晨展, 刘星雨, 张海, 曾康, 黄国强, 徐景升. 甘蔗类四跨膜蛋白ScTSPAN18与6K2互作应答SCMV侵染研究[J]. 作物学报, 2026, 52(6): 1618-1630.
[2] 陈雪燕, 何华川, 李政嘉, 董新盼, 李藕琪, 刘小云, 李丹萍, 陈志伟, 刘国霞, 吕胜源, 吴印莹, 赵振东, 曹新有, 万何平. 水培盐碱复合胁迫下‘济麦60’苗期根系有机酸分泌动态变化及其转录调控机制[J]. 作物学报, 2026, 52(6): 1859-1875.
[3] 左同鸿, 张贺翠, 曾静, 朱利泉. 甘蓝自交不亲和相关基因BoPUB3L的克隆与表达分析[J]. 作物学报, 2026, 52(6): 1698-1710.
[4] 王文辕, 燕雪嘉, 刘玉霖, 孙晓彤, 李亚楠, 唐鑫华, 石瑛. 耐弱光马铃薯品种筛选及转录因子编码基因StPIF3的克隆与功能分析[J]. 作物学报, 2026, 52(6): 1631-1645.
[5] 胡赵, 钱润, 谢丰璞, 应素平. 水稻SPX基因家族鉴定及响应磷处理的表达分析[J]. 作物学报, 2026, 52(6): 1902-1912.
[6] 韩亚鑫, 何冠华, 张小琼, 张登峰, 李永祥, 刘旭洋, 王天宇, 黎裕, 邹华文, 李春辉. 基于RNA-Seq和BSA-Seq联合分析挖掘玉米侧根密度基因资源[J]. 作物学报, 2026, 52(5): 1341-1352.
[7] 孙淑凤, 许振南, 黄嘉鑫, 翁建峰, 李新海. 玉米MAPK家族全基因组鉴定及其对拟轮枝镰孢菌感染的响应[J]. 作物学报, 2026, 52(5): 1291-1308.
[8] 田立涛, 丁宁, 王树林, 齐恩芳, 张荣, 王蕊蕊, 马利雯, 李建武, 杨江伟. 马铃薯Argonaute基因家族鉴定及晚疫病诱导表达分析[J]. 作物学报, 2026, 52(4): 1116-1126.
[9] 汪玲, 胡好, 宋家凤, 程洁蓝, 陈颖, 郑婷婷, 吕钊彦, 朱晓彪, 侯华兰. 马铃薯UDP-糖基转移酶基因StUGT52的克隆及功能验证[J]. 作物学报, 2026, 52(3): 665-676.
[10] 杨宗桃, 杨婷, 王禹童, 艾静, 李燕烨, 刘家勇, 邓军, 赵勇, 张跃彬. 甘蔗CLC基因家族鉴定与表达分析[J]. 作物学报, 2026, 52(3): 722-734.
[11] 孟成, 王哲. 玉米ZmPFK基因家族全基因组鉴定及响应胁迫表达分析[J]. 作物学报, 2026, 52(3): 764-779.
[12] 杨飚, 杜帅康, 张继旺, 石瑛, 张丽莉. 马铃薯III类POD基因家族的全基因组鉴定及其表达谱分析[J]. 作物学报, 2026, 52(2): 405-420.
[13] 余开航, 周洪斌, 罗亮扎, 王玫郦, 姜瑞梅, 董陈文华, 李仕金, 毛孝强, 陈升位. 大麦亮氨酸富集重复型类受体激酶基因HvLRR-RLK-510的克隆和表达分析[J]. 作物学报, 2026, 52(2): 421-432.
[14] 詹戈锐, 余文, 李锋, 武明珠, 徐馨, 罗朝鹏, 巫升鑫, 杨军, 张智强, 王中. 烟草NtWRKY6基因响应ABA表达及其调控多酚合成的功能研究[J]. 作物学报, 2026, 52(2): 446-458.
[15] 张力岚, 杨军, 王让剑. 基于WGCNA发掘茶树糖苷类香气前体含量性状相关的候选基因[J]. 作物学报, 2026, 52(2): 494-513.
Viewed
Full text


Abstract

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