| 同源重组快速构建百合LCHS2基因RNAi表达载体及其鉴定 |
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| 引用本文： | 化占勇,刘雅莉,徐伟荣,王跃进,张雄飞. 同源重组快速构建百合LCHS2基因RNAi表达载体及其鉴定[J]. 中国农学通报, 2010, 26(2): 38-44 |
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| 作者姓名： | 化占勇 刘雅莉 徐伟荣 王跃进 张雄飞 |
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| 作者单位： | 农业部西北园艺植物种质资源与遗传改良重点开放实验室,陕西省农业分子生物学重点实验室,西北农林科技大学园艺学院,陕西,杨凌,712100 |
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| 基金项目： | 国家自然科学基金项目"百合花色形成关键基因特异启动子功能与应用研究" |
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| 摘 要： | 为探讨CHS基因的下调表达对花朵着色的影响以及为百合观赏性状遗传改良提供技术支持,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(Genbank登录号:GQ483376)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636 bp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,测序结果显示,与原序列同源性为100%。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,经限制性内切酶XhoI、XbaI酶切鉴定获得724 bp和722 bp的片段,与预期结果一致。pH12-CHSi电击法转化农杆菌GV3101,菌液PCR鉴定,出现636 bp的目的条带,表明RNAi表达载体pH12-CHSi已成功转入农杆菌。
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| 关 键 词： | 同源重组 快速构建 LCHS2基因 RNAi 表达载体 酶切连接 |
| 收稿时间： | 2009-09-14 |
| 修稿时间： | 2009-11-01 |
Rapid Construction and Identification of RNAi Expression Vector of LCHS2 Gene from Lilium Based on the Principle of Homologous Recombination |
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| Hua Zhanyong,Liu Yali,Xu Weirong,Wang Yuejin,Zhang Xiongfei. Rapid Construction and Identification of RNAi Expression Vector of LCHS2 Gene from Lilium Based on the Principle of Homologous Recombination[J]. Chinese Agricultural Science Bulletin, 2010, 26(2): 38-44 |
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| Authors: | Hua Zhanyong Liu Yali Xu Weirong Wang Yuejin Zhang Xiongfei |
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| Affiliation: | Hua Zhanyong,Liu Yali,Xu Weirong,Wang Yuejin,Zhang Xiongfei(Key Laboratory of Northwest Horticulture Plant Germplasm , Genetic Improvement of Ministry of Agriculture of China,Key Laboratory for Molecular Biology of Agriculture of Shaanxi Province,College of Horticulture,Northwest A &F University,Yangling Shaanxi 712100) |
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| Abstract: | In order to discuss the effect of down-regulated expression of CHS gene on pigmentation of flowers and to offer technological support for genetic modification of ornamental characteristics in lilium.The Gateway technology based on homologous recombination was adopted and per the requirements of pENTR/D-TOPO vector and LCHS2 gene sequence(Genbank accession NO: GQ483376) from Oriental Lily cv 'Sorbonne',a special pair of primers were designed,the upstream of which beared 'CACC' at 5' end.An interference fragm... |
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| Keywords: | RNAi |
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