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植物研究 ›› 2011, Vol. 31 ›› Issue (6): 702-707.doi: 10.7525/j.issn.1673-5102.2011.06.011

• 论文 • 上一篇    下一篇

农杆菌介导蜡质基因WIN1转化烟草的初步研究

沙琰琰;李晓莉;史团省*;张俊;王亚杰   

  1. 郑州大学生物工程系生物多样性与生态学研究所,郑州 450001
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2011-11-20 发布日期:2011-11-20
  • 通讯作者: 史团省
  • 基金资助:
     

Preliminary Study on Agrobacterium-mediated Transformation of WIN1 Genes into Tobacco

SHA Yan-Yan;LI Xiao-Li;SHI Tuan-Sheng*;ZHANG Jun;WANG Ya-Jie   

  1. Department of Bioengineering, Zhengzhou University,Zhengzhou 450001
  • Received:1900-01-01 Revised:1900-01-01 Online:2011-11-20 Published:2011-11-20
  • Contact: SHI Tuan-Sheng
  • Supported by:
     

摘要: WIN1与蜡质代谢过程中基因的表达相关,本研究首先从野生型拟南芥花蕾中提取总RNA扩增成SHN1/WIN1,以载体PBI121为基础构建植物表达载体PBI121-SHN1/WIN1,然后利用农杆菌介导法将目的基因WIN1导入烟草k326中,在不同阶段选择四种不同培养基进行培养,其中烟草叶盘共培养培养基T1为:MS+1 mg·L-1 6-BA+0.1 mg·L-1 NAA;烟草滤菌培养基T2为:MS+1 mg·L-1 6-BA+0.1 mg·L-1 NAA+500 mg·L-1 Carb;烟草筛选培养基T3为:MS+100 mg·L-1 Kan;烟草继代培养基T4为:MS+80 mg·L-1 Kan+300 mg·L-1 Carb。本实验利用植物基因工程手段将WIN1基因整合入烟草K326中,改变烟草k326总基因序列。通过PCR、RT-PCR检测方法对转基因烟草k326一代进行鉴定得出WIN1基因成功导入烟草K326,最终获得了转基因烟草K326一代品种。本实验研究目标通过转基因技术将WIN1基因插入烟草K326,使其角质膜厚度和成分有所改变,以此来提高烟草K326抗旱、抗寒和抗病等性能,为提高烟草抗逆性特别是抗旱和抗寒性奠定基础。该项初步研究结果不仅对认识和揭示植物角质膜的结构功能具有重要理论意义,而且对应用植物基因工程技术改良和培育抗逆性强的植物优良品种和农业生产都具有重要的应用价值。

关键词: WIN1基因, 基因克隆, 烟草, 农杆菌

Abstract: The WIN1 genes are related to the process of gene expression in waxy metabolism. In this research, the total RNA was extracted from the wild type arabidopsis buds and amplified into SHN1 WIN1. The plant expression vector PBI121-SHN1/WIN1 was constructed and the WIN1 genes were inserted into tobacco k326 by using the agrobacterium-mediated technique. There were four different culture media in this research, the minimal medium was T1: MS+1 mg·L-1 6-BA+0.1 mg·L-1 NAA; the medium for filtration sterilization was T2: MS+1 mg·L-1 6-BA+0.1 mg·L-1 NAA+500 mg·L-1 Carb; the medium for screening tobacco was T3: MS+100 mg·L-1 Kan; the medium for tobacco subculture was T4: MS+80 mg·L-1 Kan+300 mg·L-1 Carb; the medium for rooting culture of tobacco was T5: MS+0.1 mg·L-1 Naa+300 mg·L-1 Carb+80 mg·L-1 Kan. In this experiment, the WIN1 genes were inserted into tobacco k326 by the plant genetic engineering to change the total gene sequences of tobacco k326. By the detection of PCR and RT-PCR, the WIN1 genes were successfully inserted into tobacco k326, and eventually the transgenic tobacco k326 were obtained. In this experiment, the WIN1 genes were inserted into tobacco k326 through the plant genetic engineering and to make the cutin membrane become thick and the composition changes are expected. Another object was to increase the drought-resistance, cold-tolerance and disease resistance of tobacco K326, therefore, the resistance of tobacco can be improved. The experiment is not only meaningful to reveal the function of plant cutin membrane structure, but also has great important application value in plant gene engineering technology and cultivate quality variety of plants and agricultural production.

Key words: WIN1 genes, gene clone, tobacco, agrobacterium

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