Ta6-SFT基因对油菜的转化及抗旱性分析

doi:10.11686/cyxb20140518

摘要/Abstract

摘要：

为了研究Ta6-SFT对油菜抗旱的影响,进行了Ta6-SFT对油菜纯系材料的遗传转化,获得经PCR、Southern杂交和Northern 斑点杂交验证的转基因植株。对7株T₁代转基因油菜进行了为期36 d的干旱胁迫,分析干旱胁迫36 d和复水后2 d各植株及野生型对照中的Ta6-SFT的转录水平表达和果聚糖含量,同时进行同时期丙二醛含量和相对电导率测定。结果表明,Ta6-SFT的转录水平表达与转基因植株体内的果聚糖含量、转基因植株的抗旱性呈正相关,表明Ta6-SFT在干旱胁迫下的表达增强了转基因植株的抗旱性。

Abstract:

Fructan is thought to be one of the important regulators involved in plant tolerance to various abiotic stresses. In this study the Triticum aestivum gene for sucrose: fructan 6-fructosyltransferase synthesis (Ta6-SFT) was transferred into Brassica napus, driven by the rd29A promoter and mediated by Agrobacterium tumefaciens. Subsequently, transgenic plants were identified using PCR, southern blot and northern dot blot tests. In order to test the effect of the Ta6-SFT gene on transgenic plants under drought stress, non-transgenic plants (control) and 7 transgenic plants (T₁ generation) were exposed to 36 days of moisture stress. Expression of Ta6-SFT was tested using a semi-quantitative RT-PCR 36 days after treatment and 2 days after re-watering. Fructan concentration and physiological traits including relative conductivity and MDA content were analyzed at the same times. The results showed Ta6-SFT expression level is positively associated with fructan content and resistance to drought stress, indicating that the Ta6-SFT gene could enhance resistance to drought in transgenic plants.

中图分类号:

S634.303.4

李淑洁,张正英. Ta6-SFT基因对油菜的转化及抗旱性分析[J]. 草业学报, 2014, 23(5): 161-167.

LI Shu-jie,ZHANG Zheng-ying. Expression of the Ta6-SFT gene in Brassica napus under drought stress[J]. Acta Prataculturae Sinica, 2014, 23(5): 161-167.

参考文献

Reference: 

[1] Van der Meerl M, Ebskamp M J M, Visser R G F, et al. Fructan as a new carbohydrate sink in transgenic potato plants[J]. Plant Cell, 1994, 6(4): 561-570.

[2] Ebstkamp M J M, Van der Meer I M, Spronk B A, et al. Accumulation of fructose polymers in transgenic tobacco[J]. BioTechnology, 1994, 12(3): 272-275.

[3] Henson C A, Livingston D P III. Characterization of a fructan exohydrolase purified from barley stems that hydrolyzes multiple fructofuranosidic linkages[J]. Plant Physiology and Biochemistry, 1998, 36: 715-720.

[4] Kawakami A, Sato Y, Yoshida M. Genetic engineering of rice capable of synthesizing fructans and enhancing chilling tolerance[J]. Journal of Experimental Botany, 2008, 59: 793-802.

[5] 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[J]. Bioscience Biotechnology and Biochemistry, 2002, 66(11): 2297-2305.

[6] Hellwege E M, Gritscher D, Willmitzer L, et al. Transgenic potato tubers accumulate high levels of 1 kestose and nystose: functional identification of a sucrose sucrose 1-fructosyltransferase of artichoke Cynara scolymus blossom discs[J]. Plant Journal, 1997, 12: 1057-1065.

[7] Hellwege E M, Raap M, Gritscher D, et al. Differences in chain-length distribution of inulin from Cynara scolymus and Helianthus tuberosus are reflected in a transient plant expression system using the respective 1 FFT cDNAs[J]. FEBS Lett, 1998, 427: 25-28.

[8] Van der Meer I M, Koops A J, Hakkert J C, et al . Cloning of fructan biosynthesis pathways of Jerusalem artichoke[J]. Plant Journal, 1998, 15: 489-500.

[9] Sévenier R, Hall R D, Van der Meer I M, et al. High level fructan accumulation in transgenic sugur beet[J]. Nature Biotechnology, 1998, 16: 843-846.

[10] Li H J, Yin H Y, Zhang X C, et al. Enhancement of drought resistance in transgenic tobacco expressing sucrose: Sucrose 1-fructosyltransferase gene from Lactuca sativa[J]. Journal of Shandong University(Natural Science), 2007, 42(1): 89-93.

[11] Zhang X Y, He J G, Sun X H. Transformation of Lolium perenne with a fructan:fructan 1-fructosyltransferase gene from Agropyron cristatum and enhancement of drought tolerance in transgenic plants[J]. Acta Prataculturae Sinica, 2011, 20: 111-118.

[12] Li S J, Wang H M, Zhang Z Y. Agrobacterium tumefaciens-based genetic transformation of brassica napus with pta and REAL-TIME PCR analysis[J]. Crops, 2010, (4): 52-55.

[13] Xue Y L. Plant physiology laboratory manual (1st Edition)[M]. Shanghai: Shanghai Science and Technology Press, 1985: 67.

[14] Li Z, Peng Y, Su X Y. Physiological responses of white clover by different leaf types associated with anti-oxidative enzyme protection and osmotic adjustment under drought stress[J]. Acta Prataculturae Sinica, 2013, 22(4): 257-263.

[15] Ye X D, Wu X L, Zhao H, et al. Altered fructan accumulation in transgenic Lolium multiflorum plants expressing a Bacillus subtilis sacB gene[J]. Plant Cell Reports, 2001, 20: 205-212.

[16] Caimi P G, McCole L M, Klein T M, et al. Fructan accumulation and sucrose metabolism in transgenic maize endosperm expressing a Bacillus amyloliquefaciens sacB gene[J]. Plant Physiology, 1996, 110: 355-363.

[17] Caimi P G, McCole L M, Klein T M, et al. Cytosolic expression of the Bacillus amyloliquefaciens SacB protein inhibits tissue development in transgenic tobacco and potato[J]. New Phytologist, 1997, 136: 19-28.

[18] Cairns A J. Fructan biosynthesis in transgenic plants[J]. Journal of Experimental Botany, 2003, 54: 549-567.

[19] Gao X, She M Y, Yin G X, et al. Isolation and Functional Determination of Fructan Biosynthesis Enzyme Encoding Gene 6-SFT from Common Wheat (Triticum aestivum L.)[J]. Science & Technology Review, 2009, 23: 70-75.

[20] Yin G X, She M Y, Gao X, et al. Cloning and characterization in structure and function of fructan biosynthesis enzymes (FBEs) gene in plant[J]. China Biotechnology, 2009, 29(2): 125-133.

[21] Bie X M, Wang K, She M Y, et al. Combinational transformation of three wheat genes encoding fructan biosynthesis enzymes confers increased fructan content and tolerance to abiotic stresses in tobacco[J]. Plant Cell Reports, 2012, 31: 2229-2238.

[22] Ye X G, Gao X, She M Y, et al. Relationship between drought tolerance and fructan biosynthesis enzymes genes and molecular biological investigation in wheat[A]. National Seminar Abstracts on Plant Molecular Breeding[C]. Beijing: China Crop Science Society会, 2009: 57.

[23] Li S J, Li J W, Zhang Z Y. Expression of Ta6-SFT gene in tobacco induced by drought stress[J]. Acta Agronomica Sinica, 2014, 40(6): 994-1001.

[24] Yu L, Liu Y H, Zhou L P, et al. 干A study on the changes of ascorbic acid and related physiological indexes in different cultivars of Zoysia under drought stress[J]. Acta Prataculturae Sinica, 2013, 22(4): 106-115.

[25] Jia X J, Dong L H, Ding C B, et al. Effects of drought stress on reactive oxygen species and their scavenging systems in Chlorophytum capense var.medio-pictum leaf[J]. Acta Prataculturae Sinica, 2013, 22(5): 248-255.

参考文献：

[1] Van der Meerl M, Ebskamp M J M, Visser R G F, et al. Fructan as a new carbohydrate sink in transgenic potato plants[J]. Plant Cell, 1994, 6（4): 561-570.

[2] Ebstkamp M J M, Van der Meer I M, Spronk B A, et al. Accumulation of fructose polymers in transgenic tobacco[J]. BioTechnology, 1994, 12（3): 272-275.

[3] Henson C A, Livingston D P III. Characterization of a fructan exohydrolase purified from barley stems that hydrolyzes multiple fructofuranosidic linkages[J]. Plant Physiology and Biochemistry, 1998, 36: 715-720.

[4] Kawakami A, Sato Y, Yoshida M. Genetic engineering of rice capable of synthesizing fructans and enhancing chilling tolerance[J]. Journal of Experimental Botany, 2008, 59: 793-802.

[5] 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[J]. Bioscience Biotechnology and Biochemistry, 2002, 66(11): 2297-2305.

[6] Hellwege E M, Gritscher D, Willmitzer L, et al. Transgenic potato tubers accumulate high levels of 1-kestose and nystose: functional identification of a sucrose sucrose 1-fructosyltransferase of artichoke Cynara scolymus blossom discs[J]. Plant Journal, 1997, 12: 1057-1065.

[7] Hellwege E M, Raap M, Gritscher D, et al. Differences in chain-length distribution of inulin from Cynara scolymus and Helianthus tuberosus are reflected in a transient plant expression system using the respective 1-FFT cDNAs[J]. FEBS Lett, 1998, 427: 25-28.

[8] Van der Meer I M, Koops A J, Hakkert J C, et al. Cloning of fructan biosynthesis pathways of Jerusalem artichoke[J]. Plant Journal, 1998, 15: 489-500.

[9] Sévenier R, Hall R D, Van der Meer I M, et al. High level fructan accumulation in transgenic sugur beet[J]. Nature Biotechnology, 1998, 16: 843-846.

[10] 李慧娟, 尹海英, 张学成, 等. 转蔗糖: 蔗糖-1-果糖基转移酶基因提高烟草的耐旱性[J]. 山东大学学报（理学版）, 2007, 42(1): 89-93.

[11] 张小芸, 何近刚, 孙学辉. 转果聚糖合成关键酶基因多年生黑麦草的获得及抗旱性的提高[J]. 草业学报, 2011, 20: 111-118.

[12] 李淑洁, 王红梅, 张正英. 半夏凝集素基因对油菜的遗传转化及REAL TIME PCR分析[J]. 作物杂志, 2010, (4): 52-55.

[13] 薛应龙. 植物生理学实验手册(第1版)[M]. 上海: 上海科学技术出版社, 1985: 67.

[14] 李州, 彭燕, 苏星源. 不同叶型白三叶抗氧化保护及渗透调节生理对干旱胁迫的响应[J]. 草业学报, 2013, 22(4): 257-263.

[15] Ye X D, Wu X L, Zhao H, et al. Altered fructan accumulation in transgenic Lolium multiflorum plants expressing a Bacillus subtilis sacB gene[J]. Plant Cell Reports, 2001, 20: 205-212.

[16] Caimi P G, McCole L M, Klein T M, et al. Fructan accumulation and sucrose metabolism in transgenic maize endosperm expressing a Bacillus amyloliquefaciens sacB gene[J]. Plant Physiology, 1996, 110: 355-363.

[17] Caimi P G, McCole L M, Klein T M, et al. Cytosolic expression of the Bacillus amyloliquefaciens SacB protein inhibits tissue development in transgenic tobacco and potato[J]. New Phytologist, 1997, 136: 19-28.

[18] Cairns A J. Fructan biosynthesis in transgenic plants[J]. Journal of Experimental Botany, 2003, 54: 549-567.

[19] 高翔, 佘茂云, 殷桂香, 等. 小麦果聚糖合成酶基因6-SFT克隆和功能验证[J]. 科技导报, 2009, 23: 70-75.

[20] 殷桂香, 佘茂云, 高翔, 等. 植物果聚糖合成酶基因克隆及特性分析[J]. 中国生物工程杂志, 2009, 29(2): 125-133.

[21] Bie X M, Wang K, She M Y, et al. Combinational transformation of three wheat genes encoding fructan biosynthesis enzymes confers increased fructan content and tolerance to abiotic stresses in tobacco[J]. Plant Cell Reports, 2012, 31: 2229-2238.

[22] 叶兴国, 高翔, 佘茂云, 等. 小麦中果聚糖合成酶基因与抗旱性关系及其分子生物学研究[A]. 全国植物分子育种研讨会摘要集[C]. 北京：中国作物学会, 2009: 57.

[23] 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分析[J]. 作物学报, 2014, 40(6): 994-1001.

[24] 俞乐, 刘拥海, 周丽萍, 等. 干旱胁迫下结缕草叶片抗坏血酸与相关生理指标变化的品种差异研究[J]. 草业学报, 2013, 22(4): 106-115.

[25] 贾学静, 董立花, 丁春邦, 等. 干旱胁迫对金心吊兰叶片活性氧及其清除系统的影响[J]. 草业学报, 2013, 22(5): 248-255.