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2025年4月5日 星期六
  2016, Vol. 24 Issue (4): 478-487    
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
低温诱导的位点特异性重组植物表达载体构建及小麦转化
王梅1,徐广博1,赵惠贤2,刘香利3
1. 西北农林科技大学
2. 西北农林科技大学生命科学学院
3. 西北农林科技大学生命学院
Construction of Low-temperature Induced Site-specific Recombinant Plant Expression Vector and Transformation of Wheat (Triticum aestivum)
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摘要 选择标记基因的剔除是转基因植物商品化种植的重要基础。为建立小麦(Triticum aestivum)低温诱导的位点特异性标记基因删除体系,本研究以小麦西农928基因组DNA为模板,通过PCR扩增获得小麦低温诱导表达wcs120启动子,序列分析表明,该序列与GenBank中序列AF031235相比有一段21 bp插入,与AY493570.1序列完全一致。以含有CinH/RS2单向位点特异性重组系统的植物表达载体pXL5513为框架,成功构建了包含有抗旱相关性基因PYL5(pyrabactin-resistance like gene 5)表达框的低温诱导的位点特异性重组植物表达载体pXL5513-fwcs-RDA;并对小麦绵阳19(M19)幼胚愈伤组织进行基因枪转化,1 745块愈伤组织经筛选分化共获得9株转基因植株,经PYL5基因特异引物PCR检测,6株为阳性植株,低温春化处理后经删除引物PCR检测,6株阳性植株初步证实成功删除了选择标记基因。本研究为利用低温诱导的位点特异性重组系统进行小麦无标记基因转化奠定了基础。
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王梅
徐广博
赵惠贤
刘香利
关键词 低温诱导wcs120启动子CinH/RS2位点特异性重组选择标记基因    
Abstract:The excision of selectable marker gene is an important foundation for transgenic crop to release into the environment or commercialized planting. In order to establish a low-temperature induced site-specific marker gene deletion system in wheat (Triticum aestivum), low-temperature induced promoter wcs120 was cloned from wheat Xinong 928 genome DNA. The sequencing result showed that the whole wcs120 promoter sequence was successfully cloned , which was the same as AY493570.1 and a insertion of 21 bp with AF031235. Using the plasmid pXL5513 which contained the CinH/RS2 system as basal vector, the final vector pXL5513-fwcs-RDA was constructed, in which the CinH recombinase gene was under the control of cold-induced promoter wcs120 and the interest gene pyrabactin-resistance like gene 5 (PYL5) was inserted outside the RS sites near the left border. All of CinH recombinase gene, selectable gene bar and reporter gene green fluorescent protein (GFP) were flanked by 2 recombination sites RS2, which would be expected to be excised after the expression of CinH recombinase. pXL5513-fwcs-RDA was transferred into wheat immature embryo calli of Mianyang 19 (M19) by particle bombardment. After selection and generation, 9 transformed plantlets were gained from 1 745 calli and 6 positive transgenic seedlings were confirmed by PCR of PYL5 gene. Induced by low temperature, all of these positive transgenic seedlings showed selectable gene deletion, which were initially confirmed by PCR. Because of the features of particle bombardment, the patterns of gene insertion were complex, the subsequent experiments were planned to identify the deletion pattern. This investigation will lay a foundation for the application of low-temperature induced site-specific recombination system in marker-free transformation on wheat.
Key wordsLow-temperature induce    wcs120 promoter    CinH/RS2 site-specific recombinant    Selectable marker gene
收稿日期: 2015-11-16      出版日期: 2016-02-24
基金资助:国家自然科学基金;国家自然科学基金;西北农林科技大学基本科研业务专项基金
通讯作者: 刘香利     E-mail: liuxianglii@163.com
引用本文:   
王梅 徐广博 赵惠贤 刘香利. 低温诱导的位点特异性重组植物表达载体构建及小麦转化[J]. , 2016, 24(4): 478-487.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2016/V24/I4/478
 
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