过量表达菊花DmDREBa基因提高转化烟草耐低温能力

doi:10.7525/j.issn.1673-5102.2016.05.013

植物研究 ›› 2016, Vol. 36 ›› Issue (5): 721-729.doi: 10.7525/j.issn.1673-5102.2016.05.013

过量表达菊花DmDREBa基因提高转化烟草耐低温能力

杨艳芳¹, 武剑², 朱凯², 刘黎卿⁴, 陈发棣³, 喻德跃²

1. 中国林业科学研究院林业研究所, 林木遗传育种国家重点实验室, 国家林业局林木培育重点实验室, 北京 100091;
2. 南京农业大学大豆研究所, 国家大豆改良中心, 作物遗传与种质创新国家重点实验室, 南京 210095;
3. 南京农业大学园艺学院观赏园艺系, 南京 210095;
4. 福建省植物生理生化重点公共实验室, 福建省亚热带植物研究所, 厦门 361009

收稿日期:2016-04-19 出版日期:2016-09-15 发布日期:2016-09-27
通讯作者: 喻德跃,E-mail:dyyu@njau.edu.cn E-mail:dyyu@njau.edu.cn
作者简介:杨艳芳(1978-),女,副研究员,现主要从事植物次生代谢工程研究。
基金资助:
国家自然科学基金项目（31300567）；中央级公益性科研院所基本科研业务费专项（CAFYBB2014QB001）

Overexpression of DmDREBa Gene Significantly Enhances Low Temperature Tolerance in Transgenic Tobacco

YANG Yan-Fang¹, WU Jian², ZHU Kai², LIU Li-Qing⁴, CHEN Fa-Di³, YU De-Yue²

1. Research Institute of Forestry, Chinese Academy of Forestry, State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation of State Forestry Administration, Beijing 100091;
2. National Center for Soybean Improvement, National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095;
3. College of Horticulture, Nanjing Agricultural University, Nanjing 210095;
4. Fujian Provincial Key Lab of Subtropic Plant Physiology and Biochemistry, Fujian Institute of Subtropical Botany, Xiamen 361009

Received:2016-04-19 Online:2016-09-15 Published:2016-09-27
Supported by:
Partly supported by National Natural Science Foundation of China(31300567);National non-profit Research Institutions of Chinese Academy of Forestry(CAFYBB2014QB001)

摘要/Abstract

摘要： 各种环境因素，如干旱、高盐、激素和低/高温等非生物胁迫对植物的生长发育造成很大影响。DREB转录因子在植物抵抗非生物胁迫中起到关键作用。本研究通过根癌农杆菌介导的叶盘转化法将菊花DmDREBa基因导入烟草中并进行了耐低温能力分析。研究利用PCR的方法鉴定出了43株转基因阳性植株。随机选取其中9株转基因植株，有7株在RNA转录水平能够表达。Southern杂交检测表明，DmDREBa基因以1~3个拷贝形式随机插入到烟草基因组中。胁迫处理结果表明，DmDREBa基因明显增强了转基因烟草抵抗低温能力。通过叶片上下表皮气孔密度检测，发现转基因烟草的蒸腾失水量远远低于对照野生型。进一步对低温胁迫下转基因烟草的丙二醛含量进行测定分析，发现转基因烟草丙二醛含量比野生型烟草低22.29%。综上结果表明，DmDREBa基因能够提高转基因烟草对低温的耐受能力，为菊花DREB转录因子的深入研究提供理论依据，并为进一步解析菊花DREB基因功能奠定基础。

关键词: 菊花, DREB, 农杆菌转化, 转基因烟草, 耐逆性

Abstract: Many environmental factors such as drought, salt stress, hormones and low/high temperature influence the growth and development of plants. It has been found that DREB transcription factors play important roles in plants against different abiotic stresses. In this study, DmDREBa(GenBank accession No.EF490996), a DREB-like gene isolated from chrysanthemum was overexpressed in tobacco plants by mediation of Agrobacterium tumefaciens under the control of the constitutive promoter CaMV 35S, and 43 transgenic tobacco plants were obtained by PCR method. 9 transgenic tobacco plants were randomly selected to perform RT-PCR investigation and 7 plants indicated RNA transcripts accumulation. The southern blot result showed that 1-3 copies of DmDREBa were randomly inserted. The tolerance of transgenic tobacco plants was increased comparing with the wild type tobacco plants under low temperature condition. The water loss of transgenic tobacco was less than wild type plants after natural transpiration. Moreover, the density of the stomata in the epidermis was observed. The result showed that the numbers of stomata in upper or lower epidermis of transgenic plants were reduced comparing to wild type tobacco. The result of t test demonstrated that the differences between the upper and lower epidermis of transgenic plants and wild type were significant. Furthermore, the content of malondialdehyde(MDA) in transgenic plants was lower than that in wild type tobacco plants at lower temperature condition, which indicated the tolerance ability of transgenic plants was increased. All these results indicated that DmDREBa improved tolerance ability of trangenic tobacco plants to the lower temperature stress. This research will provide a very useful reference for functional analysis of DREB genes in chrysanthemum in the future.

Key words: chrysanthemum, DREB, Agrobacterium-mediated transformation, transgenic tobacco(Nicotiana tabacum ‘Xantbine’), tolerance

中图分类号:

Q943.2

杨艳芳, 武剑, 朱凯, 刘黎卿, 陈发棣, 喻德跃. 过量表达菊花DmDREBa基因提高转化烟草耐低温能力[J]. 植物研究, 2016, 36(5): 721-729.

YANG Yan-Fang, WU Jian, ZHU Kai, LIU Li-Qing, CHEN Fa-Di, YU De-Yue. Overexpression of DmDREBa Gene Significantly Enhances Low Temperature Tolerance in Transgenic Tobacco[J]. Bulletin of Botanical Research, 2016, 36(5): 721-729.

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过量表达菊花DmDREBa基因提高转化烟草耐低温能力

Overexpression of DmDREBa Gene Significantly Enhances Low Temperature Tolerance in Transgenic Tobacco

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