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作物学报 ›› 2016, Vol. 42 ›› Issue (03): 389-398.doi: 10.3724/SP.J.1006.2016.00389

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大赖草6-SFT基因的克隆及其转基因烟草抗旱和抗寒性分析

贺晓岚1,王建伟2,李文旭3,陈真真1,赵继新1,武军1,王中华1,陈新宏1,*   

  1. 1 西北农林科技大学农学院 / 陕西省植物遗传工程育种重点实验室, 陕西杨凌 712100;2 凯里学院环境与生命科学学院, 贵州凯里 556011;3 河南省农业科学院小麦研究所, 河南郑州 450002
  • 收稿日期:2015-07-22 修回日期:2015-11-20 出版日期:2016-03-12 网络出版日期:2015-12-18
  • 通讯作者: 陈新宏, E-mail: cxh2089@126.com; Tel: 029-87082854
  • 基金资助:

    本研究由国家自然科学基金(31571650)项目和西北农林科技大学唐仲英育种基金资助。

Cloning of 6-SFT Gene from Leymus racemosus and Analysis of Tolerance to Drought and Cold Stresses in Transgenic Tobacco

HE Xiao-Lan1,WANG Jian-Wei2,LI Wen-Xu3,CHEN Zhen-Zhen1,ZHAO Ji-Xin1,WU Jun1,WANG Zhong-Hua1,CHEN Xin-Hong1,*   

  1. 1 Shaanxi Provincial Key Laboratory of Plant Genetic Engineering Breeding / College of Agronomy, Northwest A&F University, Yangling 712100, China; 2 College of Environment and Life Science, Kaili University, Kaili 556011, China; 3 Wheat Research Institute, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
  • Received:2015-07-22 Revised:2015-11-20 Published:2016-03-12 Published online:2015-12-18
  • Contact: 陈新宏, E-mail: cxh2089@126.com; Tel: 029-87082854
  • Supported by:

    This study was supported by the National Natural Science Foundation of China (31571650) and the Tang Zhong-Ying Breeding Funding Project of the Northwest A&F University.

摘要:

果聚糖合成酶(6-SFT)在植物抵御逆境胁迫中起重要作用。利用RACE结合RT-PCR技术从大赖草(Leymus racemosus中克隆到6-SFT基因,其完整开放阅读框为1863 bp,被命名为Lr-6-SFT (GenBank登录号KT387273),编码620个氨基酸,其推导氨基酸序列含有保守的果糖基转移酶结构域。氨基酸序列比对及进化树分析表明,大赖草6-SFT与华山新麦草、普通小麦、西尔斯山羊草和大麦6-SFT具有高度相似性。采用基因重组技术构建p1300-35SN-Lr-6-SFT表达载体,利用农杆菌介导法将该载体转入烟草品种W38中。对经过抗性筛选、PCR和RT-PCR验证的转基因植株进行抗旱和抗寒性鉴定,发现转基因植株与对照相比,抗旱和抗寒性明显增强;在逆境胁迫条件下,转基因植株的果聚糖、可溶性糖、脯氨酸含量都显著高于对照,而丙二醛的含量显著低于对照。本研究表明,Lr-6-SFT基因是典型的GH32家族成员,, 2n= 4x = 28, NsNsXmXm)其表达能够提高烟草对干旱和寒冷胁迫的抗性。

关键词: 6-SFT基因, 干旱, 寒冷, 大赖草, 生理指标, 转基因烟草

Abstract:

The fructan biosynthesis enzyme (6-SFT) plays an important role in plant responses to abiotic stresses. In this study, a full-length cDNA encoding sucrose:fructan-6-fructosyltransferase, designated as Lr-6-SFT (GenBank accession No. KT387273), was cloned from Leymus racemosus (2n = 4x = 28, NsNsXmXm) using reverse transcriptase PCR (RT-PCR) and rapid-amplification of cDNA ends (RACE) techniques. The full-length open reading frame comprises 1863 bp and encodes 620 amino acids. The predicted protein structure of the gene containes a conserved fructosyltransferase domain. Multiple sequence alignment and phylogenetic analysis showed that the Lr-6-SFT protein shared high similarity with 6-SFT proteins from Psathyrostachys huashanica, Triticum aestivum, Aegilops searsii, and Hordeum vulgare subsp. vulgare. The Lr-6-SFT gene was transferred into tobacco (Nicotiana tabacum L. cv. W38) via Agrobacterium-mediated transformation. The screened plants were tested by PCR and RT-PCR, and the transgenic tobacco plants exhibited much higher tolerance to drought and cold compared with the non-transgenic plants. Under drought and cold stresses, the Lr-6-SFT expressions were associated with the increased accumulation of stored carbohydrate and proline and the decreased malondialdehyde storage. These results suggest that Lr-6-SFT is a typical member of the glycoside hydrolase 32 (GH32) family and may be linked to enhanced tolerance to drought and cold stresses.

Key words: 6-SFT gene, Drought, Freezing, Leymus racemosus, Physiological indices, Transgenic tobacco

[1] Van Arkel J, Sévenier R, Hakkert J C, Bouwmeester H J, Koops A J, van der Meer I M. Tailor-made fructan synthesis in plants: a review. Carbohydr Polym, 2013, 93: 48–56

 [2] Hendry G A, Wallace R K. The origin, distribution, and evolutionary significance of fructans. In: Suzuki M, Chatterton N J, eds. Science and Technology of Fructans, Boca Raton, Florida, USA: The Chemical Rubber Company Press, 1993. pp 119–139

 [3] Wiemken A, Frehner M, Keller F, Wagner W. Fructan metabolism, enzymology and compartmentation: current topics in plant biochemistry and physiology. In: Proceedings of the Plant Biochemistry and Physiology Symposium. Columbia, USA: University of Missouri, 1986. pp 17–37

 [4] Hincha D K, Livingston D R, Premakumar R, Zuther E, Obel N, Cacela C, Heyer A G. Fructans from oat and rye: composition and effects on membrane stability during drying. Bioch Biophys Acta, 2007, 1768: 1611–1619

 [5] Bie X M, Wang K, She M Y, Du L, Zhang S X, Li J R, Gao X, Lin Z S, Ye X G. Combinational transformation of three wheat genes encoding fructan biosynthesis enzymes confers increased fructan content and tolerance to abiotic stresses in tobacco. Plant Cell Rep, 2012, 31: 2229–2238

 [6] Ritsema T, Joling J, Smeekens S. Patterns of fructan synthesized by onion fructan:fructan 6G-fructosyltransferase expressed in tobacco BY2 cells-is fructan:fructan 1-fructosyltransferase needed in onion? New Phytol, 2003, 160: 61–67

 [7] Sprenger N, Bortlik K, Brandt A, Boller T, Wiemken A. Purification, cloning, and functional expression of sucrose:fructan 6-fructosyltransferase, a key enzyme of fructan synthesis in barley. Proc Natl Acad Sci USA, 1995, 92: 11652–11656

 [8] Wei J Z, Chatterton N J. Fructan biosynthesis and fructosyltransferase evolution: expression of the 6-SFT (sucrose:fructan 6-fructosyltransferase) gene in crested wheatgrass (Agropyron cristatum). J Plant Physiol, 2001, 158: 1203–1213

 [9] Kawakami A, Yoshida M. Molecular characterization of sucrose:sucrose 1-fructosyltransferase and sucrose:fructan 6-fructosyltransferase associated with fructan accumulation in winter wheat during cold hardening. Biosci Biotech Biochem, 2002, 66: 2297–2305

[10] Tamura K I, Kawakami A, Sanada Y, Tase K, Komatsu T, Yoshida M. Cloning and functional analysis of a fructosyltransferase cDNA for synthesis of highly polymerized levans in timothy (Phleum pratense L.). J Exp Bot, 2009, 60: 893–905

[11] Del Viso F, Puebla A F, Fusari C M, Casabuono A C, Couto A S, Pontis H G, Hopp H E, Heinz R A. Molecular characterization of a putative sucrose:fructan 6-fructosyltransferase (6-SFT) of the cold-resistant Patagonian grass Bromus pictus associated with fructan accumulation under low temperatures. Plant & Cell Physiol, 2009, 50: 489–503

[12] He X L, Chen Z Z, Wang J W, Li W X, Zhao J X, Wu J, Wang Z H, Chen X H. A sucrose:fructan-6-fructosyltransferase (6-SFT) gene from Psathyrostachys huashanica confers abiotic stress tolerance in tobacco. Gene, 2015, 570: 239–247

[13] 高翔. 小麦族植物果聚糖合成酶基因克隆及功能验证. 中国农业科学院博士学位论文, 北京, 2010

Gao X. Cloning and Functional Analysis of Genes Encoding Fructan Biosynthesis Enzymes in Triticeae Plants. PhD Dissertation of Chinese Academy of Agricultural Sciences, Beijing, China, 2010 (in Chinese with English abstract)

[14] Del Viso F, Casabuono A C, Couto A S, Hopp H E, Puebla A F, Heinz R A. Functional characterization of a sucrose:fructan 6-fructosyltransferase of the cold-resistant grass Bromus pictus by heterelogous expression in Pichia pastoris and Nicotiana tabacum and its involvement in freezing tolerance. J Plant Physiol, 2011, 168: 493–499

[15] Li H J, Yang A F, Zhang X C, Gao F, Zhang J R. Improving freezing tolerance of transgenic tobacco expressing sucrose:sucrose 1-fructosyltransferase gene from Lactuca sativa. Plant Cell Tissue Organ Cult, 2007, 89: 37–48

[16] Wei J Z, Jerry Chatterton N, Larson S R. Expression of sucrose:fructan 6-fructosyltransferase (6-SFT) and myoinositol 1-phosphate synthase (MIPS) genes in barley (Hordeum vulgare) leaves. J Plant Physiol, 2001, 158: 635-643

[17] Tamura K, Sanada Y, Tase K, Kawakami A, Yoshida M, Yamada T. Comparative study of transgenic Brachypodium distachyon expressing sucrose:fructan 6-fructosyltransferases from wheat and timothy grass with different enzymatic properties. Planta, 2014, 239: 783–792

[18] Knipp G, Honermeier B. Effect of water stress on proline accumulation of genetically modified potatoes (Solanum tuberosum L.) generating fructans. J Plant Physiol, 2006, 163: 392–397

[19] Ruelland E, Vaultier M, Zachowski A, Hurry V. Chapter 2 cold signalling and cold acclimation in plants. Adv Bot Res, 2009, 49: 35–150

[20] Szabados L, Savouré A. Proline: a multifunctional amino acid. Trends Plant Sci, 2010, 15: 89–97

[21] Schroeven L, Lammens W, Kawakami A, Yoshida M, Van Laere A, Van den Ende W. Creating S-type characteristics in the F-type enzyme fructan:fructan 1-fructosyltransferase of Triticum aestivum L. J Exp Bot, 2009, 60: 3687–3696

[22] Chatterton N J, Harrison P A, Bennett J H, Asay K H. Carbohydrate partitioning in 185 accessions of Gramineae grown under warm and cool temperatures. J Plant Physiol, 1989, 134: 169–179

[23] Chatterton N J, Harrison P A, Thornley W R, Bennett J H. Structure of fructan oligomers in cheatgrass (Bromus tectorum L.). New Phytol, 1993, 124: 389–396

[24] Mcguire P E, Dvorak J. High salt-tolerance potential in wheatgrasses. Crop Sci, 1981, 21: 702–705

[25] Pilon-Smits E, Ebskamp M, Paul M J, Jeuken M, Weisbeek P J, Smeekens S. Improved performance of transgenic fructan-accumulating tobacco under drought stress. Plant Physiol, 1995, 107: 125–130

[26] Heyer A G, Wendenburg R. Gene cloning and functional characterization by heterologous expression of the fructosyltransferase of Aspergillus sydowi IAM 2544. Appl Environ Microbiol, 2001, 67: 363–370

[27] Chen H, Nelson R S, Sherwood J L. Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection. Biotechniques, 1994, 16: 664–668

[28] Schuler M A, Zielinski R E. Transformation of leaf discs with Agrobacterium. In: Gelvin S B, Schilperoort R A, Verma D P S, eds. Methods in Plant Molecular Biology. San Diego: Academic Press, 1989. pp 145–156

[29] Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning (Third Edn). New York: Cold Spring Harbor Laboratory Press, 1989

[30] Fales F W. The assimilation and degradation of carbohydrates by yeast cells. J Biol Chem, 1951, 193: 113–124

[31] Bates L S, Waldren R P, Teare I D. Rapid determination of free proline for water-stress studies. Plant and Soil, 1973, 39: 205–207

[32] Lasseur B, Schroeven L, Lammens W, Le Roy K, Spangenberg G, Manduzio H, Vergauwen R, Lothier J, Prud' Homme M P, Van den Ende W. Transforming a fructan:fructan 6G-fructosyltransferase from perennial ryegrass into a sucrose:sucrose 1-fructosyltransferase. Plant Physiol, 2009, 149: 327–339

[33] 岳爱琴, 李昂, 毛新国, 昌小平, 李润植, 贾继增, 景蕊莲. 小麦果聚糖合成酶基因6-SFT-A单核苷酸多态性分析及其定位. 中国农业科学, 2011, 44: 2216–2224

Yue A Q, Li A, Mao X G, Chang X P, Li R Z, Jia J Z, Jing R L. Single nucleotide polymorphism and mapping of 6-SFT-A gene responsible for fructan biosynthesis in common wheat. Sci Agric Sin, 2011, 44: 2216–2224 (in Chinese with English abstract)

[34] Gao X, She M Y, Yin G X, Yu Y, Qiao W H, Du L P, Ye X G. Cloning and characterization of genes coding for fructan biosynthesis enzymes (FBEs) in triticeae plants. Agric Sci China, 2010, 9: 313–324

[35] 李淑洁, 李静雯, 张正英. Ta6-SFT在烟草中的逆境诱导型表达及抗旱性. 作物学报, 2014, 40: 994–1001

Li S J,Li J W,Zhang Z Y. Expression of Ta6-SFT Gene in tobacco induced by drought stress. Acta Agron Sin, 2014, 40: 994–1001 (in Chinese with English abstract)

[36] 文明, 卜利伟, 罗紫韵, 王佳伟, 董芬, 黄雪松. 大蒜蔗糖﹕蔗糖1-果糖基转移酶(1-SST)的酶学特征. 中国农业科学, 2015, 48: 334–342

Wen M, Bu L W, Luo Z Y, Wang J W, Dong F, Huang X S. Characteristics of sucrose:sucrose 1-fructosyltransferase in garlic. Sci Agric Sin, 2015, 48: 334–342 (in Chinese with English abstract)

[37] Tamura K, Sanada Y, Tase K, Yoshida M. Fructan metabolism and expression of genes coding fructan metabolic enzymes during cold acclimation and overwintering in timothy (Phleum pratense). J Plant Physiol, 2014, 171: 951–958

[38] 许欢欢, 康健, 梁明祥. 植物果聚糖的代谢途径及其在植物抗逆中的功能研究进展. 植物学报, 2014, 49: 209–220

Xu H H, Kang J, Liang M X. Research advances in the metabolism of fructan in plant stress resistance. Chin Bull Bot, 2014, 49: 209–220 (in Chinese with English abstract)

[39] Venkateswarlu B, Shanker A K, Shanker C, Maheswari M. Crop Stress and its Management: Perspectives and Strategies. Springer Netherlands, 2012

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