小麦胚乳14-3-3基因的克隆及其重组蛋白的原核表达

doi:10.3864/j.issn.0578-1752.2012.10.021

摘要/Abstract

摘要： 【目的】克隆小麦籽粒胚乳14-3-3基因，并进行体外表达，为进一步研究其对籽粒生长发育的调控作用奠定基础。【方法】根据已有同源基因保守序列，设计插入限制性酶切位点的特异性扩增引物，采用RT-PCR方法扩增发育的小麦胚乳14-3-3基因，克隆测序后转入表达载体，在大肠杆菌中进行表达，并进行纯化。【结果】从开花后灌浆13—15 d的小麦品种济麦22籽粒胚乳中克隆到1个14-3-3基因，序列分析表明为非ε型，含1个777 bp的开放阅读框，编码蛋白259 aa，分子量约29 kD。核苷酸序列分析表明与小麦、水稻、玉米、大麦、大豆等主要农作物和模式植物拟南芥的14-3-3基因有较高的同源性，最高达98%，编码蛋白氨基酸长度也一致（260 aa左右）；在大肠杆菌中高效表达的重组蛋白约为30 kD，分子量大小与根据核苷酸序列推导的编码蛋白一致。从基因序列的同源性、编码蛋白的氨基酸长度、表达蛋白的分子量大小分析都说明克隆到的基因为14-3-3基因，并准确插入表达载体，得到了高效正确表达。将克隆的基因插入pET29c载体，热激转化大肠杆菌BL21-CodonPlus(DE3)-RP，得到了高效表达，但主要以包涵体形式（80%）存在。对重组蛋白进行了纯化，可溶性重组蛋白利用S-蛋白琼脂糖树脂得到纯化的蛋白，包涵体重组蛋白经变性溶解、复性后，也利用S-蛋白琼脂糖树脂得到了高度纯化的重组蛋白。【结论】利用RT-PCR技术从发育的小麦胚乳中克隆到1个14-3-3基因，并在大肠杆菌中得到了高效表达，重组蛋白经过纯化得到了纯度较高的活性蛋白。

关键词: 小麦, 14-3-3蛋白, 基因克隆, 重组蛋白, 原核表达

Abstract: 【Objective】 This research was conducted to clone 14-3-3 genes from developing wheat endosperm and express their recombinant proteins in Escherichia coli aiming to investigate their functions in wheat development.【Method】Specific primers with restriction enzymes cut site were designed according to conserved sequence of homologous genes registered in NCBI. The target genes were cloned by RT-PCR from filling wheat grain. Then the cloned genes were inserted into expressing vectors after confirmation by sequencing and multiple alignments. The recombinant protein was expressed in E. coli and purified for further research. 【Result】 A 14-3-3 gene was amplified from developing endosperm of 13-15 d after anthesis of bread wheat cultivar Jimai22 and inserted into Top plasmid vector then transformed into E. coli strain DH5α by heat shock. The cloned gene was sequenced after the recombinant plasmid was extracted. The results of sequencing analysis showed that the gene belongs to non-ε group and contained an open reading frame of 777 bp in length encoding a protein with 259 aa with predicted molecular weight about 29 kD. According to multiple alignments using DNAMAN program, the gene was highly homologous to other 14-3-3 genes from main crops such as wheat, rice, maize, barley, soybean and model plant Arabidopsis with the maximum homology of 98%, as well as their encoding proteins. Furthermore, the heterologous protein with molecular weight about 30 kD expressed in E. coli was coincident with predicted size based on deduced amino acid sequence. All results suggested that the cloned gene is a 14-3-3 gene and it was correctly inserted into the vector and expressed heterologously. The cloned gene was inserted into expressing vector pET29c possessing a S-tag specific bounding to S-protein agarose. The recombinant vector was transformed into E. coli strain BL21-CodonPlus (DE3)-RP supplied with additional rare codes to improve heterologous expression. The recombinant protein was expressed at very high level, however, existed mainly as an insoluble inclusion body (about 80%) after extraction by BugBuster Protein Extraction Reagent. The soluble fusion protein was purified directly by bounding to S-protein agarose followed by washing out of non-specific bounding proteins and other impurities, while the inclusion body should be dissolved in 8 mol L-1 urea and refolded firstly. 【Conclusion】A 14-3-3 gene was cloned from developing wheat endosperm through RT-PCR and heterologously expressed at high level in E. coli. Purified 14-3-3 recombinant protein with bioactivity was harvested for further study by purification with S-protein agarose.

Key words: wheat (Triticum aestivum L.), 14-3-3 protein, gene clone, recombinant protein, heterologous expression

戴双, 李豪圣, 程敦公, 刘爱峰, 曹新有, 刘建军, 宋健民. 小麦胚乳14-3-3基因的克隆及其重组蛋白的原核表达[J]. 中国农业科学, 2012, 45(10): 2076-2084.

DAI Shuang, LI Hao-Sheng, CHENG Dun-Gong, LIU Ai-Feng, CAO Xin-You, LIU Jian-Jun, SONG Jian-Min. Cloning of a 14-3-3 Gene from Developing Wheat Endosperm and Expression of its Recombinant Protein in Escherichia coli[J]. Scientia Agricultura Sinica, 2012, 45(10): 2076-2084.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2012.10.021

https://www.chinaagrisci.com/CN/Y2012/V45/I10/2076

参考文献

[1]Aitken A. 14-3-3 proteins: A historic overview. Seminars in Cancer Biology, 2006, 16: 162-172.

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

[3]Chevalier D, Morris E R, Walker J C. 14-3-3 and FHA domains mediate phosphoprotein interactions. Annual Review of Plant Biology, 2009, 60: 67-91.

[4]Denison F C, Paul A L, Zupanska A K, Ferl R J. 14-3-3 proteins in plant physiology. Seminars in Cell and Developmental Biology, 2011, 22(7): 720-727.

[5]Wilker E W, van Vugt M A T M, Artim S A, Huang P H, Petersen C P, Christian Reinhardt H, Feng Y, Sharp P A, Sonenberg N, White F M, Yaffe M B. 14-3-3σ controls mitotic translation to facilitate cytokinesis. Nature, 2007, 446: 329-332.

[6]Sehnke P C, Chung H J, Wu K, Ferl R J. Regulation of starch accumulation by granule-associated plant 14-3-3 proteins. Proceedings of the National Academy of Sciences of the United States of America, 2001, 98: 765-770.

[7]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 Physiology, 2008, 146: 1878-1891.

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

[9]Pozuelo Rubio M, Geraghty K M, Wong B H C, 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. Biochemical Journal, 2004, 379: 395-408.

[10]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 Physiology, 2007, 143(2): 670-683.

[11]Lu G, DeLisle A J, de Vetten N C, Ferl R J. Brain proteins in plants: An Arabidopsis homolog to neurotransmitter pathway activators in part of a DNA binding complex. Proceedings of the National Academy of Sciences of the United States of America, 1992, 89: 11490-11494.

[12]Ferl R J, Lu G, Bowen B W. Evolutionary implications of the family of 14-3-3 brain protein homologs in Arabidopsis thaliana. Genetica, 1994, 92(2): 129-138.

[13]Wu K, Rooney M F, Ferl R J. The Arabidopsis 14-3-3 multigene family. Plant Physiology, 1997, 114: 1421-1431.

[14]Rosenquist M, Alsterfjord M, Larsson C, Sommarin M. Data mining the Arabidopsis genome reveals fifteen 14-3-3 genes. Expression is demonstrated for two out of five novel genes. Plant Physiology, 2001, 127(1): 142-149.

[15]Sehnke P C, Laughner B, Cardasis H, Powell D, Ferl R J. Exposed loop domains of complexed 14-3-3 proteins contribute to structural diversity and functional specificity. Plant Physiology, 2006, 140(2): 647-660.

[16]Cooper B, Clarke J D, Budworth P, Kreps J, Hutchison D, Park S, Guimil S, Dunn M, Luginbühl P, Ellero C, Goff S A, Glazebrook J. A network of rice genes associated with stress response and seed development. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100(8): 4945-4950.

[17]Kikuchi S, Satoh K, Nagata T, Kawagashira N, Doi K, Kishimoto N, Yazaki J, Ishikawa M, Yamada H, Ooka H, Hotta I, Kojima K, Namiki T, Ohneda E, Yahagi W, Suzuki K, Li C J, Ohtsuki K, Shishiki T, Otomo Y, Murakami K, Iida Y, Sugano S, Fujimura T, Suzuki Y, Tsunoda Y, Kurosaki T, Kodama T, Masuda H, Kobayashi M, Xie Q, Lu M, Narikawa R, Sugiyama A, Mizuno K, Yokomizo S, Niikura J, Ikeda R, Ishibiki J, Kawamata M, Yoshimura A, Miura J, Kusumegi T, Oka M, Ryu R, Ueda M, Matsubara K, Kawai J, Carninci P, Adachi J, Aizawa K, Arakawa T, Fukuda S, Hara A, Hashizume W, Hayatsu N, Imotani K, Ishii Y, Itoh M, Kagawa I, Kondo S, Konno H, Miyazaki A, Osato N, Ota Y, Saito R, Sasaki D, Sato K, Shibata K, Shinagawa A, Shiraki T, Yoshino M, Hayashizaki Y, Yasunishi A. Collection, mapping, and annotation of over 28 000 cDNA clones from japonica rice. Science, 2003, 301(5631): 376-379.

[18]Li X, Dhaubhadel S. Soybean 14-3-3 gene family: Identification and molecular characterization. Planta, 2011, 233: 569-582.

[19]Umezawa T, Sakurai T, Totoki Y, Toyoda A, Seki M, Ishiwata A, Akiyama K, Kurotani A, Yoshida T, Mochida K, Kasuga M, Todaka D, Maruyama K, Nakashima K, Enju A, Mizukado S, Ahmed S, Yoshiwara K, Harada K, Tsubokura Y, Hayashi M, Sato S, Anai T, Ishimoto M, Funatsuki H, Teraishi M, Osaki M, Shinano T, Akashi R, Sakaki Y, Yamaguchi-Shinozaki K, Shinozaki K. Sequencing and analysis of approximately 40 000 soybean cDNA clones from a full-length-enriched cDNA library. DNA Research, 2008, 15(6): 333-346.

[20]Brandt J, Thordal-Christensen H, Vad K, Gregersen P L, Collinge D B. A pathogen-induced gene of barley encodes a protein showing high similarity to a protein kinase regulator. The Plant Journal, 1992, 2(5): 815-820.

[21]Schoonheim P J, Sinnige M P, Casaretto J A, Veiga H, Bunney T D, Quatrano R S, de Boer A H. 14-3-3 adaptor proteins are intermediates in ABA signal transduction during barley seed germination. The Plant Journal, 2007, 49(2): 289-301.

[22]de Vetten N C, Lu G, Ferl R J. A maize protein associated with the G-box binding complex has homology to brain regulatory proteins. The Plant Cell, 1992, 4: 1295-1307.

[23]Alexandrov N N, Brover V V, Freidin S, Troukhan M E, Tatarinova T V, Zhang H, Swaller T J, Lu Y P, Bouck J, Flavell R B, Feldmann K A. Insights into corn genes derived from large-scale cDNA sequencing. Plant Molecular Biology, 2009, 69(1/2): 179-194.

[24]Ikeda Y, Koizumi N, Kusano T, Sano H. Specific binding of a 14-3-3 protein to autophosphorylated WPK4, an SNF1-related wheat protein kinase, and to WPK4-phosphorylated nitrate reductase. The Journal of Biological Chemistry, 2000, 275(41): 31695-31700.

[25]Yao Y, Ni Z, Zhang Y, Chen Y, Ding Y, Han Z, Liu Z, Sun Q. Identification of differentially expressed genes in leaf and root between wheat hybrid and its parental inbreds using PCR-based cDNA subtraction. Plant Molecular Biology, 2005, 58(3): 367-384.

[26]Wang C, Ma Q H, Lin Z B, He P, Liu J Y. Cloning and characterization of a cDNA encoding 14-3-3 protein with leaf and stem-specific expression from wheat. Mitochondrial DNA, 2008, 19(2): 130-136.

[27]Esposito D, Chatterjee D K. Enhancement of soluble protein expression through the use of fusion tags. Current Opinion in Biotechnology, 2006, 17: 353-358.

[28]Waugh D S. Making the most of affinity tags. Trends in Biotechnology, 2005, 23(6): 316-320.

[29]Sambrook J, Russell D W. Molecular Cloning: A Laboratory Mannual: Third Edition. New York: Cold Spring Harbor Laboratory Press, 2001.

[30]宋健民, 戴双, 李豪圣, 刘爱峰, 程敦公, 楚秀生, Tetlow I J, Emes M J. 小麦胚乳14-3-3蛋白的表达及其与淀粉体淀粉合成酶的互作. 作物学报, 2009, 35(8): 1445-1450.

Song J M, Dai S, Li H S, Liu A F, Cheng D G, Chu X S, Tetlow I J, Emes M J. Expression of a wheat endosperm 14-3-3 protein and its interactions with starch biosynthetic enzymes in amyloplasts. Acta Agronomica Sinica, 2009, 35(8): 1445-1450. (in Chinese)

[31]Lambeck I, Chi J C, Krizowski S, Mueller S, Mehlmer N, Teige M, Fischer K, Schwarz G. Kinetic analysis of 14-3-3-inhibited Arabidopsis thaliana nitrate reductase. Biochemistry, 2010, 49(37): 8177-8186.