过量表达蔗糖转运蛋白基因增强转基因小麦的耐旱性

doi:10.3864/j.issn.0578-1752.2015.08.02

摘要/Abstract

摘要： 【目的】创制过量表达TaSUT1A的转基因小麦，分析TaSUT1A在转基因小麦中的遗传及其对干旱胁迫的应答反应，选育抗旱的转基因小麦新种质。【方法】采用基因重组技术构建了TaSUT1A表达载体，利用基因枪介导法将该载体转入小麦品种科农199，通过Bialaphos筛选、转化植株基因组DNA PCR验证获得转基因T₀植株；利用RT-PCR检测TaSUT1A在转基因T₃植株中的表达情况，在此基础上，对3个转基因系的T₄转基因植株进行抗旱性鉴定和抗旱相关生理指标分析，验证其抗旱能力。【结果】经PCR检测和RT-PCR验证，获得了转TaSUT1A小麦阳性植株，与非转基因对照相比，20%PEG胁迫处理显著诱导了转基因株系根叶组织中目标基因TaSUT1A的上调表达。抗旱鉴定和抗性生理分析显示，在20%PEG胁迫处理下，转基因株系的萌发率比非转基因对照平均提高了32.8%，显著高于非转基因对照，并促进初生根的萌发和生长，初生根长和胚芽鞘长比非转基因对照平均增加了81.72%和170.77%；在20%PEG胁迫处理下，转基因植株叶组织中的蔗糖和可溶性糖平均提高了42.95%和36.56%，根中蔗糖和可溶性糖平均提高了58.01%和43.01%，均显著高于非转基因对照植株；与未胁迫处理相比，20%PEG胁迫处理后非转基因植株叶中的SOD活性由105.4 U·g^-1FW升高到139.1 U·g^-1FW，而转基因植株的活性由107.7—115.3 U·g^-1FW提高到168.2—211.6 U·g^-1FW，显著高于非转基因对照，同时，转基因小麦株系的MDA的产生较非转基因对照平均降低了37.47%，显著减少了MDA的产生。【结论】TaSUT1A在参与植物的逆境应答反应机制中具有重要作用，促进逆境胁迫中小麦的萌发和生长，超量表达TaSUT1A可显著提高转基因小麦的耐旱能力。

关键词: 普通小麦, 蔗糖转运蛋白, 转基因, 干旱胁迫, 可溶性糖

Abstract: 【Objective】 Drought is one of the most important constraints resulting in large yield losses and limiting the average yield increase of wheat in China. The aim of this study is to develop and select stable TaSUT1A transgenic wheat lines with resistance to drought.【Method】The open-reading-frame sequence of TaSUT1A was synthesized and used to construct the gene transformation vector pUBI::cas-TaSUT1A, in which TaSUT1A gene was driven by maize ubiquitin promoter and should be highly expressed in monocot plants. Particle bombardment method was used to introduce TaSUT1A into wheat cultivar Kenong 199. After bialaphos screening, DNA PCR and RT-PCR methods were used to detect the presence and transcript levels of TaSUT1A in the transgenic wheat plants of T₀-T₄ generations. Subsequently, the resistance to water stress of transgenic wheat plants and non-transgenic wheat Kenong 199 at young seedling-stage was evaluated. Further, their determination of physiologic index related to abiotic stress was finished and the potential of improving abiotic stress tolerance in plant was elucidated. 【Result】The results indicated that the introduced TaSUT1A gene was stably inherited, and 220% PEG treatment could significantly induce the expression of TaSUT1A in root and leaf tissues of three transgenic wheat lines. The average germination rate of transgenic wheat lines was 85.53%, which was more higher than those of non-transgenic controls, and the average celoeptile and primary root length significantly higher than the non-transgenic plants. The results showed that the transgenic wheat significantly enhanced seed germination in 20% PEG treatment, as indicated by enhancement of the growth of celoeptile and primary root. Further, three pure lines with higher TaSUT1A expression were selected to elucidate physiologic index related to abiotic stress. In detail, in 20% PEG treatment, the transgenic lines overexpressing TaSUT1A increased sucrose and total soluble sugar content in leaf and root, with the average increase value of 42.95% and 36.56% in leaf, and 58.01% and 43.01% in root respectively, which was significantly higher than the non-transgenic plants. After treatment under water stress condition, the MDA content of transgenic plants only increased by 6.12 nmol·g^-1FW, however, the MAD content of the non-transgenic plants increased by 19.05 nmol·g^-1FW. Meanwhile, the transgenic plant showed better SOD activity, with the average increase value of 44.7 U·g^-1FW, which was significantly higher than the non-transgenic plants. 【Conclusion】It was concluded that TaSUT1A plays an important role in response to drought stress in plants, overexpression of TaSUT1A can improve significantly tolerance to drought stress in transgenic wheat plants.

Key words: common wheat, sucrose transporter, transgenic, drought stress, soluble sugar

胡梦芸，李辉，庞建周，刘茜，张颖君，孙丽静. 过量表达蔗糖转运蛋白基因增强转基因小麦的耐旱性[J]. 中国农业科学, 2015, 48(8): 1473-1483.

HU Meng-yun, LI Hui, PANG Jian-zhou, LIU Qian, ZHANG Ying-jun, SUN Li-jing. Overexpression of Sucrose Transporter (TaSUT1A) Improves Drought Tolerance in Transgenic Wheat[J]. Scientia Agricultura Sinica, 2015, 48(8): 1473-1483.

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参考文献

[1] 山仑. 生物节水研究现状及展望//香山科学会议第267次学术讨论会集. 北京: 香山科技会议, 2005: 3-14.

Shan L. Advances in biological water-saving research: Challenge and perspectives//A summary of the Academic Conference on 267th XiangShan-Science Conferences. Beijing: XiangShan- Science Conferences, 2005: 3-14. (in Chinese)

[2] Zhang J X, Klueva N Y, Wang Z, Wu R, Ho T H D, Nguyen H T. Genetic engineering for abiotic stress resistance in crop plants. In Vitro Cellular & Developmental Biology Plant, 2000, 36(2): 108-114.

[3] Ashraf M, Athar H R, Harris P J C, Kwon T R. Some prospective strategies for improving crop salt tolerance. Advances in Agronomy, 2008, 97: 45-110.

[4] Rizhsky L, Liang H, Shuman J, Shulaev V, Davletova S, Mittler R. When defense pathways collide. The response of Arabidopsis to a combination of drought and heat stress. Plant Physiology, 2004, 134: 1683-1696.

[5] Gupta A K, Kaur N. Sugar signaling and gene expression in relation to carbohydrate metabolism under abiotic stresses in plants. Journal of Biosciences, 2005, 30: 761-776.

[6] Rosa M, Prado C, Podazza G, Interdonato R, Juan A G, Hilal M, Prado F E. Soluble sugars-Metabolism, sensing and abiotic stress: A complex network in the life of plants. Plant Signaling Behavior, 2009, 4(5): 388-393.

[7] Ivan C. Involvement of soluble sugars in reactive oxygen species balance and responses to oxidative stress in plants. Journal of Experimental Botany, 2006, 57(3): 449-459.

[8] Rolland F. Sugar sensing and signalling in plants, conserved and novel mechanisms. Annual Review of Plant Biology, 2006, 57: 675-709.

[9] Silvaa E N, Luiz F S S, Viégasb R A, Silveiraa J A G. The role of organic and inorganic solutes in the osmotic adjustment of drought- stressed Jatropha curcas plants. Environmental and Experimental Botany, 2010, 69(3): 279-285.

[10] Sivakumar P, Sharmila P, Jain V, Saradhi P P. Sugars have potential to curtail oxygenase activity of Rubisco. Biochemical and Biophysical Research Communications, 2002, 298: 247-250.

[11] Wingenter K. Increased activity of the vacuolar monosaccharide transporter TMT1 alters cellular sugar partitioning, sugar signalling and seed yield in Arabidopsis. Plant Physiology, 2010, 13: 1104-1110.

[12] Yamada K, Osakabe Y, Mizoi J, Nakashima K, Fujita Y, Shinozaki K, Yamaguchi-Shinozaki K. Functional analysis of an Arabidopsis thaliana abiotic stress-inducible facilitated diffusion transporter for monosaccharides. Journal of Biological Chemistry, 2010, 285(2): 1138-1146.

[13] 马小龙, 刘颖慧, 袁祖丽, 石云素, 宋燕春, 王天宇, 黎裕. 玉米蔗糖转运蛋白基因ZmERD6 cDNAs的克隆与逆境条件下的表达. 作物学报, 2009, 35(8): 1410-1417.

Ma X L, Liu Y H, Yuan Z L, Shi Y S, Song Y C, Wang T Y, Li Y. Cloning of cDNAs for a novel sugar transporter gene, ZmERD6, from maize and its expression analysis under abiotic stresses. Acta Agronomica Sinica, 2009, 35(8): 1410-1417. (in Chinese)

[14] Aoki N, Scofield G, Wang X D, Patrick J, Offler C, Furbank R T. Expression and localisation analysis of the wheat sucrose transporter TaSUT1 in vegetative tissues. Planta, 2004, 219: 176-184.

[15] Aoki N, Scofield G N, Wang X D, Offler C E, Patrick J W, Furbank R T. Pathway of sugar transport in germinating wheat seeds. Plant Physiology, 2006, 141: 1255-1263.

[16] 梁晓芳, 于振文. 施钾时期对冬小麦旗叶光合特性和籽粒淀粉积累的影响. 应用生态学报, 2004, 15(8): 1349-1352.

Liang X F, Yu Z W. Effect of potassium application stage on photosynthetic characteristics of winter wheat flag leaves and on starch accumulation in wheat grains. Chinese Journal of Applied Ecology, 2004, 15(8): 1349-1352. (in Chinese)

[17] 周玉梅, 韩士杰, 张军辉, 邹春静, 王琛瑞. CO₂浓度升高对长白山三种树木幼苗叶碳水化合物和氮含量的影响. 应用生态学报, 2002 13(6): 663-666.

Zhou Y M, Han S J, Zhang J H, Zou C J, Wang S R. Effect of elevated CO₂ concentration on carbohydrate and nitrogen contents in seedlings foliage of three tree species in Changbai Mountain. Chinese Journal of Applied Ecology, 2002, 13(6): 663-666. (in Chinese)

[18] Wang R, Chen S, Ma H, Liu L, Li H, Weng H, Hao Z, Yang S. Genotypic differences in antioxidative stress and salt tolerance of three poplars under salt stress. Frontiers of Forestry in China, 2006, 1: 82-88.

[19] 赵世杰. 植物生理学实验指导. 北京: 中国农业科学技术出版社, 2004.

Zhao S J. Plant Physiology Experimental Guidance. Beijing: China Agricultural Science and Technology Press, 2004. (in Chinese)

[20] 兰巨生. 农作物综合抗旱性评价方法的研究. 西北农业学报, 1998, 7(3): 85-87.

Lan J S. The research of identification drought resistance and its evaluation method. Acta Agriculturae Boreali-Occidentalis Sinica, 1998, 7(3): 85-87. (in Chinese)

[21] Keunen E, Peshev D, Vangronsveld J, Ende W V D, Cuypers A. Plant sugars are crucial players in the oxidative challenge during abiotic stress: Extending the traditional concept. Plant Cell and Environment, 2013, 36: 1242-1255.

[22] Eveland A L, Jackson D P. Sugars, signalling, and plant development. Journal of Experimental Botany, 2012, 63: 3367-3377.

[23] 杨晓杰, 刘传亮, 张朝军, 武芝霞, 张雪妍, 刘坤, 房卫平, 李付广. 不同转化方法获得的转基因棉花外源基因拷贝数分析. 农业生物技术学报, 2011, 19(2): 221-229.

Yang X J, Liu C L, Zhang Z J, Wu Z X, Zhang X Y, Liu K, Fang W P, Li F G. Comparative analysis of exogenous gene copy numbers in transgenic cotton transformed by different methods. Journal of Agricultural Biotechnology, 2011, 19(2): 221-229. (in Chinese)

[24] 王玮, 邹琦. 胚芽鞘长度作为冬小麦抗旱性鉴定指标的研究. 作物学报, 1997, 23(4): 459-467.

Wang W, Zou Q. Studies on coleoptile length as criterion of appraising drought resistance in wheat. Acta Agronomica Sinica, 1997, 23(4): 459-467. (in Chinese)

[25] 吴永成, 周顺利, 王志敏, 赵长星. 冬小麦初生根系与抗旱性及产量关系的初步研究. 青岛农业大学学报: 自然科学版, 2008, 25(3): 168-170.

Wu Y C, Zhou S L, Wang Z M, Zhao C X. Preliminary study on the relationship between primary root system and drought resistance and yield of winter wheat. Journal of Qingdao Agricultural University: Natural Science, 2008, 25(3): 168-170. (in Chinese)

[26] Gill P K, Sharma A D, Singh P, Bhullar S S. Effect of various abiotic stresses on the growth soluble sugars and water relations of sorghum seedlings grown in light and darkness. Journal of Plant Physiology, 2001, 27: 72-84.

[27] Gibson S I. Control of plant development and gene expression by sugar signaling. Current Opinion Plant Biology, 2005, 8: 93-102.

[28] 贺鸿雁, 孙存华, 杜伟, 李扬. PEG6000胁迫对花生幼苗渗透调节物质的影响. 中国油料作物学报, 2006, 28(1): 76-78.

He H Y, Sun C H, Du W, Li Y. Effects of PEG6000 osmtic stress on osmolytes of peanut seedling. Chinese Journal of Oil Crop Sciences, 2006, 28(1): 76-78. (in Chinese)

[29] 胡梦芸, 李辉, 张颖君, 刘茜. 水分胁迫下葡萄糖对小麦幼苗光合作用和相关生理特性的影响. 作物学报, 2009, 35(4): 724-732.

Hu M Y, Li H, Zhang Y J, Liu Q. Photosynthesis and related physiological characteristics affected by exogenous glucose in wheat seedlings under water stress. Acta Agronomica Sinica, 2009, 35(4): 724-732. (in Chinese)

[30] Hu M Y, Shi Z G, Zhang Z B, Zhang Y J, Li H. Effects of exogenous glucose on seed germination and antioxidant capacity in wheat seedlings under salt stress. Plant Growth Regulation, 2012, 68: 177-188.

[31] Siringam K, Juntawong N, Chaum S, Boriboonkaset T, Kirdmanee C. Salt tolerance enhancement in indica rice (Oryza sativa L.) seedlings using exogenous sucrose supplementation. Plant Omics, 2012, 5(1): 52-59.

[32] 赵莹, 杨克军, 赵长江, 李佐同, 王玉凤, 付健, 郭亮, 李文胜. 外源糖调控玉米光合系统和活性氧代谢缓解盐胁迫. 中国农业科学, 2014, 47(20): 3962-3972.

Zhao Y, Yang K J, Zhao C J, Li Z T, Wang Y F, Fu J, Guo L, Li W S. Alleviation of the adverse effects of salt stress by regulating photosynthetic system and active oxygen metabolism in maize seedlings. Scientia Agricultura Sinica, 2014, 47(20): 3962-3972. (in Chinese)

[33] Bolouri-Moghaddam R M, Roy K L, Li X, Rolland F, Van den Ende W. Sugar signalling and antioxidant network connections in plant cells. The FEBS Journal, 2010, 277: 2022-2037.

[34] Garg A K, Kim J K, Owens T G, Ranwala A P, Do Choi Y, Kochian L V, Wu R J. Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses. Proceedings of the National Academy Sciences of the United States of America, 2002, 99: 15898-15903.

[35] Sulmon C, Gouesbet C, Amrani A E, Couée S. Sugar-induced tolerance to the herbicide atrazine in Arabidopsis seedlings involves activation of oxidative and xenobiotic stress responses. Plant Cell Reports, 2006, 25: 489-498.

[36] Price J, Laxmi A, Martin S K S, Jang J C. Global transcription pro?ling reveals multiple sugar signal transduction mechanisms in Arabidopsis. The Plant Cell, 2004, 16: 2128-2150.