碱茅6-磷酸海藻糖合成酶基因的克隆及其耐逆性分析

doi:10.11686/cyxb20150113

草业学报 ›› 2015, Vol. 24 ›› Issue (1): 99-106.DOI: 10.11686/cyxb20150113

碱茅6-磷酸海藻糖合成酶基因的克隆及其耐逆性分析

李莹^{1, 2}, 柳参奎^{1*, *}

1.东北林业大学盐碱地生物资源环境研究中心,黑龙江哈尔滨 150040;
2.黑龙江八一农垦大学寒地作物种质改良与栽培重点实验室,黑龙江大庆 163319

收稿日期:2013-12-13 出版日期:2015-01-20 发布日期:2015-01-20
通讯作者: shenkuiliu@nefu.edu.cn
作者简介:李莹(1979-),女,黑龙江哈尔滨人,讲师,博士。 E-mail:LY7966@163.com
基金资助:
黑龙江省寒地作物种质改良与栽培重点实验开放课题资助项目(CGIC201204),教育部创新团队发展计划(IRT13053),黑龙江省自然科学基金项目(C201406),中央高校基本科研业务费专项资金(2572014BA20),863项目(2013AA102701-7)和哈尔滨市自然科学基金(2008RFQXN007)资助

Cloning of a TPS gene and analysis of its function in stress tolerance in Puccinellia tenuiflora

LI Ying^{1, 2}, LIU Shenkui^{1, *}

1.Alkali Soil Natural Environmental Science Center, Northeast Forestry University, Harbin 150040, China;
2.Key Laboratory of Crop Germplasm Improvement and Cultivation in Cold Regions, Heilongjiang Bayi Agricultural University, Daqing 163319, China

Received:2013-12-13 Online:2015-01-20 Published:2015-01-20

摘要/Abstract

摘要： 从碱茅酵母cDNA文库中经过NaCl、NaHCO₃筛选,均得到长为1153 bp碱茅6-磷酸海藻糖合成酶基因(PutTPS)片段。通过3'End cDNA amplification方法获得基因缺失的3'序列,该基因全长3358 bp,编码882个氨基酸。氨基酸序列比较结果表明,PutTPS氨基酸序列与水稻、拟南芥、玉米等多种高等植物的TPS蛋白的同源性高达60%90%。利用Northern blot技术,研究该基因的组织表达模式及NaCl、NaHCO₃胁迫处理下基因的表达模式变化;同时对转pYES₂- PutTPS基因酵母菌株进行盐、碱、氧化胁迫、渗透胁迫处理,观察其在逆境下的生存表现。结果表明,PutTPS具有组织表达特异性,其中在根和花中表达量最大;NaCl、NaHCO₃会诱导PutTPS 基因在根和叶中的上调表达;同时重组酵母菌株对盐碱、氧化、渗透胁迫及干旱等逆境的适应能力显著增强。以上研究结果表明,碱茅PutTPS基因与逆境之间具有一定的应答关系,并在植物适应环境逆境过程中起着重要的作用。

Abstract: In this study, a 6-trehalose phosphate synthase gene (TPS) fragment, 1153 bp in length, was isolated through NaCl and NaHCO₃ screening from Puccinellia tenuiflora yeast cDNA library. A 3'-end cDNA amplification technique was used to clone PutTPS. The full-length cDNA is 3358 bp containing an open reading frame (ORF) of 882 amino acids. Multiple alignment analysis based on amino acids of TPS proteins of rice, Arabidopsis, maize, and other higher plants revealed that the amino acid homologies were about 60% to 90%.The tissue expression patterns of P. tenuiflora PutTPS and the expression under NaCl and NaHCO₃ stress were further investigated by northern blot. Also the recombinant yeast INVScI (pYES₂-PutTPS) and control INVScI (pYES₂) were subjected to salt, drought, osmotic and oxidative stresses. PutTPS was highly expressed in roots and flowers; in roots and leaves, NaCl and NaHCO₃ induced up-regulated expression of PutTPS genes and the recombinant yeast cells showed higher stress resistance than control cells. It was also found that the TPS from alkali grass contributes to salt, drought, osmotic and oxidative resistance.The present study indicated that there was a relationship between PutTPS expression and multiple stresses, and PutTPS may play an important role for P. tenuiflora during adaption to environmental abiotic stress.

李莹, 柳参奎. 碱茅6-磷酸海藻糖合成酶基因的克隆及其耐逆性分析[J]. 草业学报, 2015, 24(1): 99-106.

LI Ying, LIU Shenkui. Cloning of a TPS gene and analysis of its function in stress tolerance in Puccinellia tenuiflora[J]. Acta Prataculturae Sinica, 2015, 24(1): 99-106.

参考文献

[1] Elbein A D, Pan Y T, Pastuszak I, et al . New insights on trehalose: a multifunctional molecule. Glycobiology, 2003, 13(4): 17-27.
[2] Alvarez-Peral F J, Zaragoza O, Pedreno Y, et al . Protective role of trehalose during severe oxidative stress caused by hydrogen peroxide and the adaptive oxidative stress response in Candida albicans . Microbiology, 2002, 148(8): 2599-2606.
[3] Crowe J H, Crowe L M, Chapman D. Preservation of membranes in anhydrobiotic organisms: the role of trehalose. Science, 1984, 223(4637): 701-703.
[4] De Virgilio C, Hottiger T, Dominguez J, et al . The role of trehalose synthesis for the acquisition of thermotolerance in yeast. I. Genetic evidence that trehalose is a thermoprotectant. European Journal of Biochemistry/FEBS, 1994, 219(1-2): 179-186.
[5] Garcia A B, Engler J, Iyer S, et al . Effects of osmoprotectants upon NaCl stress in rice. Plant Physiology, 1997, 115(1): 159-169.
[6] Lillie S H, Pringle J R. Reserve carbohydrate metabolism in Saccharomyces cerevisiae : responses to nutrient limitation. Journal of Bacteriology, 1980, 143(3): 1384-1394.
[7] Giaever H M, Styrvold O B, Kaasen I, et al . Biochemical and genetic characterization of osmoregulatory trehalose synthesis in Escherichia coli . Journal of Bacteriology, 1988, 170(6): 2841-2849.
[8] Reinders A, Burckert N, Hohmann Surcker T N, et al . Structural analysis of the subunits of the trehalose-6-phosphate synthase/phosphatase complex in Saccharomyces cerevisiae and their function during heat shock. Molecular Microbiology, 1997, 24(4): 687-695.
[9] Siebers B, Tjaden B, Michalke K, et al . Reconstruction of the central carbohydrate metabolism of Thermoproteus tenax by use of genomic and biochemical data. Journal of Bacteriology, 2004, 186(7): 2179-2194.
[10] Blazquez M A, Santos E, Flores C L, et al . Isolation and molecular characterization of the Arabidopsis TPS1 gene, encoding trehalose-6-phosphate synthase. The Plant Journal: for Cell and Molecular Biology, 1998, 13(5): 685-689.
[11] Vogel G, Aeschbacher R A, Muller J, et al . Trehalose-6-phosphate phosphatases from Arabidopsis thaliana : identification by functional complementation of the yeast tps2 mutant. The Plant Journal: for Cell and Molecular Biology, 1998, 13(5): 673-683.
[12] Kosmas S A, Argyrokastritis A, Loukas M G, et al . Isolation and characterization of drought-related trehalose 6-phosphate-synthase gene from cultivated cotton ( Gossypium hirsutum L.). Planta, 2006, 223(2): 329-339.
[13] Pramanik M H, Imai R. Functional identification of a trehalose 6-phosphate phosphatase gene that is involved in transient induction of trehalose biosynthesis during chilling stress in rice. Plant Molecular Biology, 2005, 58(6): 751-762.
[14] Shima S, Matsui H, Tahara S, et al . Biochemical characterization of rice trehalose-6-phosphate phosphatases supports distinctive functions of these plant enzymes. The FEBS Journal, 2007, 274(5): 1192-1201.
[15] Wang Y J, Hao Y J, Zhang Z G, et al . Isolation of trehalose-6-phosphate phosphatase gene from tobacco and its functional analysis in yeast cells. Journal of Plant Physiology, 2005, 162(2): 215-223.
[16] Zentella R, Mascorro-Gallardo J O, Van Dijck P, et al . A Selaginella lepidophylla trehalose-6-phosphate synthase complements growth and stress-tolerance defects in a yeast tps1 mutant. Plant Physiology, 1999, 119(4): 1473-1482.
[17] Leyman B, Van Dijck P, Thevelein J M. An unexpected plethora of trehalose biosynthesis genes in Arabidopsis thaliana . Trends in Plant Science, 2001, 6(11): 510-513.
[18] Xie X, Kirby J, Keasling J D. Functional characterization of four sesquiterpene synthases from Ricinus communis (castor bean). Phytochemistry, 2012, 78: 20-28.
[19] Zang B, Li H, Li W, et al . Analysis of trehalose-6-phosphate synthase (TPS) gene family suggests the formation of TPS complexes in rice. Plant Molecular Biology, 2011, 76(6): 507-522.
[20] Zhuang X, Kollner T G, Zhao N, et al . Dynamic evolution of herbivore-induced sesquiterpene biosynthesis in sorghum and related grass crops. The Plant Journal: for Cell and Molecular Biology, 2012, 69(1): 70-80.
[21] Vandesteene L, Ramon M, Le Roy K, et al . A single active trehalose-6-P synthase (TPS) and a family of putative regulatory TPS-like proteins in Arabidopsis . Molecular Plant, 2010, 3(2): 406-419.
[22] Li H W, Zang B S, Deng X W, et al . Overexpression of the trehalose-6-phosphate synthase gene OsTPS1 enhances abiotic stress tolerance in rice. Planta, 2011, 234(5): 1007-1018.
[23] Avonce N, Leyman B, Mascorro-Gallardo J O, et al . The Arabidopsis trehalose-6-P synthase AtTPS1 gene is a regulator of glucose, abscisic acid, and stress signaling. Plant Physiology, 2004, 136(3): 3649-3659.
[24] Peng Y H, Zhu Y F, Mao Y Q, et al . Alkali grass resists salt stress through high [K + ] and an endodermis barrier to Na + . Journal of Experimental Botany, 2004, 55(398): 939-949.
[25] Sambrook J R, David W. Molecular Cloning: A Laboratory Manual 3rd ed[M]. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2001.
[26] Scotto-Lavino E, Du G, Frohman M A. Scotto-lavino EDu GFrohman M A 3' end cDNA amplification using classic RACE. Nature Protocols, 2006, 1(6): 2742-2745.
[27] Reshkin S J, Cassano G, Womersley C, et al . Preservation of glucose transport and enzyme activity in fish intestinal brush border and basolateral membrane vesicles. Journal of Experimental Biology, 1988, 140: 123-135.
[28] Kong L F, Zhang J Y, Liu Z P, et al . Cloning of a S-adenosyl methionine synthetase gene from Cleistogenes songorica and its expression under drought stress. Acta Prataculturae Sinica, 2013, 22(1): 268-275.
[29] Ma Y, Guo L Q, Zhang S F, et al . Solute accumulation and distribution traits of an alkali resistant forage plant kochia sieversiana and physiological contribution of organic acid under salt and alkali stresses. Acta Prataculturae Sinica, 2013, 22(1): 193-200.
[30] Li X Y, Lin J X, Li X J, et al . Growth adaptation and Na + and K + metabolism responses of Leymus chinensis seedlings under salt and alkali stresses. Acta Prataculturae Sinica, 2013, 22(1): 201-209.
[31] Vanlaere A. Trehalose, reserve and/or stress metabolite. FEMS Microbiol Lett, 1989, 63(3): 201-210.
[32] Jang I C, Oh S J, Seo J S, et al . Expression of a bifunctional fusion of the escherichia coli genes for trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in transgenic rice plants increases trehalose accumulation and abiotic stress tolerance without stunting growth. Plant Physiology, 2003, 131(2): 516-524.
[28] 孔令芳, 张吉宇, 刘志鹏, 等. 无芒隐子草SAMS1基因的克隆及干旱胁迫下的表达分析. 草业学报, 2013, 22(1): 268-275.
[29] 麻莹, 郭立泉, 张淑芳,等. 盐碱胁迫下抗碱牧草碱地肤溶质积累、分布特点及有机酸的生理贡献. 草业学报, 2013, 22(1):193-200.
[30] 李晓宇, 蔺吉祥, 李秀军, 等. 羊草苗期对盐碱胁迫的生长适应及Na + 、K + 代谢响应. 草业学报, 2013, 22(1): 201-209.

碱茅6-磷酸海藻糖合成酶基因的克隆及其耐逆性分析

Cloning of a TPS gene and analysis of its function in stress tolerance in Puccinellia tenuiflora

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