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中国农学通报

中国农学通报 ›› 2010, Vol. 26 ›› Issue (2): 38-44.

所属专题： 生物技术

• 生物技术科学 • 上一篇    下一篇

同源重组快速构建百合LCHS2基因RNAi表达载体及其鉴定

化占勇,刘雅莉,徐伟荣,王跃进,张雄飞   

  • 收稿日期:2009-09-14 修回日期:2009-11-01 出版日期:2010-01-20 发布日期:2010-01-20
  • 基金资助:

    国家自然科学基金项目《花色形成关键基因特异启动子功能与应用研究》

Rapid Construction of RNAi Expression Vector of LCHS2 Gene from Lilium Based on the Principle of Homologous Recombination

  • Received:2009-09-14 Revised:2009-11-01 Online:2010-01-20 Published:2010-01-20

PDF (PC)

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摘要：

摘要：利用基于同源重组原理的Gateway技术，根据课题组从东方百合‘索邦’分离的LCHS2基因序列和pENTR/D TOPO载体要求，设计上游5'端添加'CACC'的一对特异引物，PCR扩增获得636bp的干扰片段。通过TOPO克隆，将该片段克隆入载体pENTR/D-TOPO 构建入门克隆载体pENTR/D-CHS。再经过LR反应使pENTR/D-CHS上的干扰片段替换掉目标载体pHellsgate12上的ccdB片段，快速构建了包含LCHS2基因干扰片段的RNAi表达载体pH12-CHSi。pH12-CHSi电击法转化农杆菌GV3101，为下一步转化矮牵牛，展开CHS基因的下调表达对花色影响的研究提供了一定的基础。

关键词: 澳洲坚果, 澳洲坚果, Ikaika(333), 产量, 品质, 抗风性

Abstract:

Abstract：With the Gateway technology based on homologous recombination and for the request of pENTR TOPO vector and LCHS2 gene sequence from Oriental Lily cv 'Sorbonne', a special pair of primers were designed, the upstream of which beared 'CACC' at 5' end. An interference fragment of 636bp was obtained by PCR amplification with the primers above and cloned into pENTR/D-TOPO to form pENTR/D-CHS via TOPO cloning. Then the ccdB fragment on destination vector pHellsgate12 was replaced by the interference fragment on pENTR/D-CHS via LR reaction to form RNAi expression vector pH12-CHSi, which was transformed into Agrobacterium strain GV3101 by electroporation. The finished work provides foundation for petunia transformation and research on the effect of down-regulated expression of CHS gene on pigmentation of flowers.