垫状卷柏海藻糖-6-磷酸合成酶基因的克隆及功能分析
收稿日期: 2009-10-09
修回日期: 2009-12-02
网络出版日期: 2010-05-15
基金资助
国家科技重大专项(编号：2009ZX08003-012B)和国家自然科学基金项目(编号：30671309)资助
Cloning and functional analysis of trehalose -6-phosphate synthase gene from Selaginella pulvinata
Received date: 2009-10-09
Revised date: 2009-12-02
Online published: 2010-05-15
海藻糖-6-磷酸合成酶(Trehalose-6-phosphate synthse, TPS)是植物海藻糖合成途径的关键酶, 在旱生卷柏等复苏植物对逆境胁迫应答中起重要作用。文章以我国特有旱生植物垫状卷柏(Selaginella pulvinata)为材料, 采用同源扩增与RACE技术相结合的方法克隆了海藻糖-6-磷酸合成酶基因SpTPS1, cDNA全长3 223 bp, 包括一个2 790 bp的开放阅读框, 推导的氨基酸序列与模式物种的海藻糖-6-磷酸合成酶具有较高的序列相似性, 催化活性中心保守位点基本一致。酵母功能互补实验证明, 用SpTPS1基因开放阅读框转化的海藻糖合成酶基因突变(tps1△)酵母菌株, 可恢复在以葡萄糖作为唯一碳源培养基上的生长, 说明垫状卷柏海藻糖-6-磷酸合成酶基因SpTPS1的编码蛋白具有生物活性, 可应用于植物抗逆性的转基因改良。
关键词： 垫状卷柏; 海藻糖-6-磷酸合成酶; 基因克隆; 功能
林荆，付凤玲，蒋伟，牟禹，雍太明，李晚忱 . 垫状卷柏海藻糖-6-磷酸合成酶基因的克隆及功能分析[J]. 遗传, 2010 , 32(5) : 498 -504 . DOI: 10.3724/SP.J.1005.2010.00498
Trehalose-6-phosphate synthase, a key enzyme in trehalose synthesis pathway of plant, plays an important role in response to abiotec stress in xerophilous rock lily and other resurrection plants. In this study, homologous amplification and RACE technique were used to clone gene SpTPS1 for trehalose-6-phosphate synthase from Selaginella pulvinata, which is an endemic xerophilous plant in China. The full-length cDNA is 3 223 bp long, containing an open reading frame (ORF) of 2 790 bp. Protein sequence comparison showed that the pupative amino acid sequence of this ORF shares high similarity to trehalose-6-phosphate synthases of mode speciese, especially at the conserved sites of catalytic activity centers. Yeast functinal complementation test showed that trehalose-6-phosphate synthase mutant (tps1△), transformed by the ORF of SpTPS1 gene, can restore growth on the mediam supplemented with glucose as a sole carbon source. This result indicated that SpTPS1 of S. pulvinata encodes for an activative protein and is hopeful to be applied in transgeneic improvement of abiotic stress tolerance in plant.
[1] 王关林, 方宏筠主编. 植物基因工程(第二版). 北京: 科学出版社, 2002, 61–73.
[2] Colaco C, Kampinga J, Roser B. Amorphous stability and trehalose. Science, 1995, 268(5212): 788.
[3] Crowe JH, Crowe LM, Chapman D. Preservation of mem-branes in anhydrobiotic organisms: the role of trehalose. Science, 1984, 223(4637): 701–703.
[4] Kaasen I, Falkenberg P, Styrvold OB, strom AR. Molecu-lar cloning and physical mapping of the otsB genes, which encode the osmoregulatory trehalose pathway of Es-cherichia coli: evidence that transcription is activated by katF (AppR). J Bactreiol, 1992, 174(3): 889–898.
[5] Bell W, Klaassen P, Ohnacker M, Boller T, Herweijer M, Schoppink P, Van der Zee P, Wiemken A. Characterization of the 56-kDa subunit of yeast trehalose-6-phosphate syn-thase and cloning of its gene reveal its identity with the product of CIF1, a regulator of carbon catabolite inactiva-tion. Eur J Biochem, 1992, 209(3): 951–959.
[6] Zentella R, Mascorro-Gallardo JO, Van Dijck P, Folch-Mallol J, Bonini B, Van Vaeck C, Gaxiola R, Co-varrubias AA, Nieto-Sotelo J, Thevelein JM, Iturriaga G. A Selaginella lepidophylla trehalose-6-phosphate synthase complements growth and stress-tolerance defects in a yeast tps1 mutant. Plant Physiol, 1999, 119(4): 1473–1482.
[7] Serrano R, Villalba JM. Expression and localization of plant membrane proteins in Saccharomyces. Methods Cell Biol, 1995, 50: 481–496.
[8] Christianson TW, Sikorski RS, Dante M, Shero JH, Hieter P. Multifunctional yeast high-copy number shuttle vectors. Gene, 1992, 110(1): 119–122.
[9] Elble R. A simple and efficient procedure for transforma-tion of yeasts. BioTechniques, 1992, 13(1): 18–20.
[10] Gibson RP, Turkenburg JP, Charnock SJ, Lloyd R, Davies GJ. Insights into trehalose synthesis provided by the structure of the retaining glucosyltransferase OtsA. Chem Biol, 2002, 9(12): 1337–1346.
[11] Boume Y, Henrissat B. Glycoside hydrolases and glyco-syltransferases: families and functional modules. Curr Opin Struct Biol, 2001, 11(5): 593–600.
[12] Van Aelst L, Hohmann S, Bulaya B, de Koning W, Sierk-stra L, Neves MJ, Luyten K, Alijo R, Ramos J, Coccetti P. Molecular cloning of a gene involved in glucose sensing in the yeast Saccharomyces cerevissiae. Mol Microbiol, 1993, 8(5): 927–943.
[13] Thevelein JM, Hohmann S. Trehalose synthase: guard to the gate of glycolysis in yeast? Trends Biochem Sci, 1995, 20(1): 3–10.
[14] Blazquez MA, Lagunas R, Gancedo C, Gancedo IM. Tre-halose-6-phosphate, a new regulator of yeast glycolysis that inhibits hexokinases. FEBS Lett, 1993, 329(1–2): 51–54.
[15] Blazquez MA, Santos E, Flores CL, Martinez Zapater JM, Salinas J, Gancedo C. Isolation and characterization of the Arobidopsis TPS1 gene. Plant J, 1998, 13(5): 685–689.
[16] Tarczynski MC, Jensen RG, Bohnert HJ. Expression of a bacterial mtlD gene in transgenic tobacco leads to produc-tion and accumulation of mannitol. Proc Natl Acad Sci USA, 1992, 89(7): 2600–2604.
[17] Holmstrom KO, Mantyla E, Welin B, Mandal A, Palva ET, Tunnela OE, Londesborough J. Drought tolerance in to-bacco. Nature, 1996, 379(6567): 683–684.
[18] Goddijn OJ, Verwoerd TC, Voogd E, Krutwagen RW, de Graaf PT, van Dun K, Poels J, Ponstein AS, Damm B, Pen J. Inhibition of trehalose activity enhances trehalose accumulation in trans-genic plants. Plant Physiol, 1997, 113(1): 181–190.
[19] Romero C, Belles JM, Vaya JL, Serrano R, Culianez-Macia FA. Expression of the yeast trehalose-6-phosphate synthase gene in transgenic tobacco plants: pleiotropic phenotypes include drought tolerance. Planta, 1997, 201(3): 293–297.
[20] Pilon-Smits E
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