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作物学报 ›› 2014, Vol. 40 ›› Issue (09): 1572-1578.doi: 10.3724/SP.J.1006.2014.01572

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

结缕草CBF基因的同源克隆及其转基因拟南芥的抗寒性验证

冯勋伟1,2,才宏伟1   

  1. 1中国农业大学农学与生物技术学院, 北京 100193; 2国家知识产权局专利局审查协作北京中心, 北京 100190
  • 收稿日期:2014-03-10 修回日期:2014-06-16 出版日期:2014-09-12 网络出版日期:2014-07-09
  • 通讯作者: 才宏伟, E-mail: caihw@cau.edu.cn, Tel: 010-62734224
  • 基金资助:

    本研究由国家自然科学基金项目(30771372)资助。

Cloning of Zoysiagrass CBF Gene and Validation of Cold Tolerance in Transgenic Arabidopsis

FENG Xun-Wei1,2,CAI Hong-Wei1   

  1. 1 College of Agriculture and Biotechnology, China Agricultural University, Beijing 100190, China; 2 Patent Examination Cooperation Center of the Patent Office, SIPO, Beijing 100190, China
  • Received:2014-03-10 Revised:2014-06-16 Published:2014-09-12 Published online:2014-07-09
  • Contact: 才宏伟, E-mail: caihw@cau.edu.cn, Tel: 010-62734224

摘要:

结缕草是优良的暖季型草坪草之一, 主要用于亚热带和热带地区的草坪种植。抗冷性是结缕草栽培范围的限制因子。本研究以日本最北部原产的结缕草品系为材料, 根据其他植物的已知的抗寒基因CBF序列, 通过同源克隆的方法获得结缕草中相对应的同源基因ZjCBF;根据和其他已报告的CBF序列的比对结果, 我们确定ZjCBF基因属于CBF转录因子家族基因中CBF1型基因。利用半定量PCR和实时定量PCR分析该基因在寒冷条件下的表达情况, 发现ZjCBF基因受冷胁迫的诱导, 4处理6 h时表达量最高。在此基础上, 本研究构建了该基因的过表达载体, 并将其转化到拟南芥中, 通过低温冷处理实验发现, 不论是否经过冷驯化, 转基因ZjCBF的植株由于ZjCBF的过量表达都要比野生型植株抗寒性强

关键词: 结缕草, 耐寒性, CBF转录因子, 同源克隆, 转基因拟南芥

Abstract:

Zoysiagrass is recognized as an excellent warm-season turfgrass and mainly used in subtropical and tropical regions. Cold stress is a major constraint factor for the cultivation of zoysiagrass. In this study, according to the sequences of cold tolerance gene CBF had been reported in other plant species, we cloned the corresponding homologous of the ZjCBF gene by homology cloning method in Zoysia japonica using a material originated from the most northern area of Japan. Based on the alignment results compared with other reported CBF genes, we found the ZjCBF gene belongs to the CBF1 familiy. By semi-quantitative PCR and Real-time quantitative PCR, we analyzed the expression level of the ZjCBF gene in the cold condition and found that ZjCBF was induced by cold stress, and the ZjCBF expression reached peak after six hours at 4°C treatment. In addition, we also constructed ZjCBF over expression vector and generated transgenic Arabidopsis plants, with better cold tolerance than the wild-type, whether through cold acclimation or not.

Key words: Zoysiagrass, Cold tolerance, CBF transcription factor, Homologous cloning, Transgenic Arabidopsis

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