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

草业学报 ›› 2011, Vol. 20 ›› Issue (1): 111-118.

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

张小芸¹,何近刚^1,2,孙学辉¹,吴金霞^1*

1.中国农业科学院生物技术研究所,北京100081;
2.河北省农林科学院遗传生理研究所,河北石家庄 050051

出版日期:2011-02-22 发布日期:2011-02-22
通讯作者: E-mail:jinxia@caas.net.cn
作者简介:张小芸(1985-),女,山西吕梁人,硕士。
基金资助:
国家 973计划项目 (2007CB108900)和863(2006AA10Z124)项目资助。

Transformation of Lolium perenne with a fructan:fructan 1-fructosyltransferase gene from
Agropyron cristatum and enhancement of drought tolerance in transgenic plants

ZHANG Xiao-yun¹, HE Jin-gang^1,2, SUN Xue-hui¹, WU Jin-xia¹

1.Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China;

2.Institute of Genetics and Physiology, Hebei Academy of Agricultural
and Forestry Sciences, Shijiazhuang 050051, China

Online:2011-02-22 Published:2011-02-22

摘要/Abstract

摘要： 以多年生黑麦草(品种卡特)胚性愈伤组织为转化受体,利用农杆菌介导的遗传转化方法将冰草果聚糖:果聚糖-1-果糖基转移酶基因(Ac1-FFT)导入黑麦草中,对再生植株喷洒basta溶液和PCR法检测,共获得18个阳性株系,RT-PCR结果表明,该基因在转基因黑麦草中正常表达。转基因黑麦草株系中的可溶性总糖含量和果聚糖含量明显高于对照植株,耐旱性提高,干旱胁迫6 d时其相对含水量和叶绿素含量明显高于对照植株,且下降速度慢,但其电解质渗漏率和丙二醛含量显著低于对照植株,复水后很快复原,而对照植株无法恢复,说明转基因植株中由于干旱处理发生的损伤是可逆的,而对照植株中的损伤是非可逆的。以上结果表明,转基因黑麦草中Ac1-FFT的表达及果聚糖合成可能是其耐旱性提高的最重要原因。

Abstract: Ac1-FFT (fructan:fructan 1-fructosyltransferase) cloned from Agropyron cristatum was transformed into embryogenic callus lines of Lolium perenne using an Agrobacterium mediated method. Transgenic plants were analyzed by basta spraying and PCR amplification. In total, 18 independent transgenic lines were obtained with significantly improved drought tolerance. RT-PCR analysis showed that Ac1-FFT expressed strongly in transgenic L. perenne plants but not in control plants. Water soluble carbohydrate and fructan contents of Ac1-FFT transgenic L. perenne were significantly higher than those in control plants. Relative water content and chlorophyll content were significantly higher, while electrolyte leakage and malondialdehyde (MDA) content were significantly lower in the Ac1-FFT transgenic L. perenne than in control plants for 6 days after drought treatment. Relative water content, chlorophyll content, electrolyte leakage and MDA content were restored to their original levels after rewatering in Ac1-FFT transgenic plants but not in control plants. This suggests that the damage to Ac1-FFT transgenic plants was reversible but it was not in control plants. Ac1-FFT expression and fructan biosynthesis may play an important role in improving drought tolerance of Ac1-FFT transgenic L. perenne.

中图分类号:

张小芸,何近刚,孙学辉,吴金霞. 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(1): 111-118.

ZHANG Xiao-yun, HE Jin-gang, SUN Xue-hui, WU Jin-xia. 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(1): 111-118.

参考文献

Zhao H, Bughrara S S. Isolation and characterization of cold-regulated transcriptional activator LpCBF3 gene from perennial ryegrass (Lolium perenne L.). Molecular Genetics and Genomics, 2008, 279: 585-594.
Shivendra B, Yidong R, Jonathan P. A high throughput Agrobacterium tumefaciens-mediated transformation method for functional genomics of perennial ryegrass (Lolium perenne L.). Plant Cell Reports, 2006, 25: 651-659.
Hiroshi H, Kanazawa A, Kawakami A, et al. Transgenic perennial ryegrass plants expressing wheat fructosyltransferase genes accumulate increased amounts of fructan and acquire increased tolerance on a cellular level to freezing. Plant Science, 2004, 167(4): 861-868.
Yancey P H. Living with water stress: Evolution of osmolyto system. Science, 1982, 217: 1214-1222.
梁哲, 姜三杰, 未丽, 等. 三叶草基因工程研究进展. 草业学报, 2009, 18(2): 205-211.
Hendry G F. Evolutionary origins and natural fractions of fructans: A climatological, biogeographic and mechanistic appraisal. New Phytologist, 1993, 123: 3-14.
Turk S C H J, Smeekens S. Genetic modification of plant carbohydrate metabolism. In: Chopra V L, Malik V S, Bhat S R. Applied Plant, Biotechnology. InC, USA: Science Publishers, 1999: 71-100.
Ebstamp M J M, Van der Meer I M, Spronk B A, et al. Accumulation of fructose polymers in transgenic tobacco. Biotechnology, 1994, 12: 272-275.
Pilon-Smits E A H, Ebsramp M J M, Jerken M J M, et al. Improved performance of transgenic fructan-accumulating tobacco under drought stress. Plant Physiology, 1995, 107: 125-130.
Knipp G, Honermeier B. Effect of water stress on proline accumulation of genetically modified potatoes (Solanum tuberosum L.) generating fructans. Plant Physiology, 2006, 163: 1-6.
Li H J, Yang A F, Zhang X C, et al. Improving freezing tolerance of transgenic tobacco expressing sucrose: Sucrose 1-fructosyltransferase from Lactuca sativa. Plant Cell, Tissue and Organ Culture, 2007, 89: 37-48.
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. 山东大学学报, 2007, 42(1): 89-94.
吴金霞, 陈彦龙, 何近刚, 等. 生物技术在牧草品质改良中的应用. 草业学报, 2007, 16(1): 1-9.
成善汉, 谢从华, 柳俊. 高等植物果聚糖研究进展. 植物学通报, 2002, 19(3): 280-289.
Ye X D, Wu X L, Zhao H, et al. Altered fructan accumulation in transgenic Lolium multiflorum plants expressing a Bacillus subtilis sacB gene. Plant Cell Reports, 2001, 20: 205-212.
王奇丽, 何近刚, 陈彦龙, 等. 农杆菌介导多年生黑麦草转化体系的建立. 中国农业科技导报, 2009, 11(2): 119-123.
徐春波, 米福贵, 王勇, 等. 影响冰草成熟胚组织培养再生体系频率的因素. 草业学报, 2009, 18(1): 80-85.
Chomczynski P. A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques, 1993, 15(3): 532-534, 536-537.
王晶英, 敖红, 张杰, 等. 植物生理生化实验技术与原理. 哈尔滨: 东北林业大学出版社, 2003.
李得孝, 郭月霞, 员海燕, 等. 玉米叶绿素含量测定方法研究. 中国农学通报, 2005, 21(6): 153-155.
刘燕琼, 黄雪松. 大蒜中果聚糖的测定. 食品与发酵工业, 2005, 31(8): 84-86.
张远兵, 刘爱荣, 张雪平. 13个冷季型草坪草品种在蚌埠地区的适应性研究. 草业科学, 2009, 26(4): 127-133.
万里强, 石永红, 李向林, 等. 高温干旱胁迫下三个多年生黑麦草品种叶绿体和线粒体超微结构的变化. 草业学报, 2009, 18(1): 25-31.
王军辉, 查学强, 罗建平, 等. 干旱胁迫对玉米幼苗脂质过氧化作用及保护酶活性的影响. 安徽农业科学, 2006, 34(15): 3568-3569, 3571.
Hincha D K, Hellwege E M, Heyer A G, et al. Plant fructan stabilize phosphatidycholine liposomes during freeze-drying. European Journal of Biochemistry, 2000, 267: 535-540.

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