| 优化反向PCR法分离转基因油菜外源T-DNA侧翼序列的研究 |
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| 引用本文： | 杨坤,吴学龙,朗春秀,陈锦清.优化反向PCR法分离转基因油菜外源T-DNA侧翼序列的研究[J].安徽农业科学,2010,38(10):5002-5005. |
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| 作者姓名： | 杨坤 吴学龙 朗春秀 陈锦清 |
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| 作者单位： | 浙江师范大学化学与生命科学学院,浙江,金华,321004;浙江省农业科学院病毒学与生物技术研究所,浙江省植物基因工程代谢重点实验室,浙江,杭州,310021;浙江省农业科学院病毒学与生物技术研究所,浙江省植物基因工程代谢重点实验室,浙江,杭州,310021 |
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| 基金项目： | 国家"863"计划项目，浙江省自然科学基金项目 |
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| 摘 要： | 目的]优化反向PCR（IPCR）条件分离油菜转基因外源T-DNA的侧翼序列。方法]以单拷贝的转基因油菜FS4为研究材料,用改良CTAB法提取转基因油菜总DNA,并进行酶切、纯化、自连接,再通过两轮巢式PCR扩增,进行序列对比。结果]两轮巢式PCR扩增得到1个大小为4.0kb的片段,其序列与油菜数据库中BH652424和BZ044084两序列同源性分别高达97.8%和63.4%。根据序列比对结果设计特异引物对进行PCR验证的结果,也证明了FS4转基因油菜中T-DNA的确插入在该位点。结论]优化后的IPCR条件能成功分离油菜转基因外源T-DNA的侧翼序列。
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| 关 键 词： | 反向PCR 侧翼序列 改良CTAB法 转基因油菜 |
Study on the Isolation of the Flanking Sequences Adjacent to Transgenic T-DNA in Brassica napus Genome by an Improved Inverse PCR Method |
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| YANG Kun et al.Study on the Isolation of the Flanking Sequences Adjacent to Transgenic T-DNA in Brassica napus Genome by an Improved Inverse PCR Method[J].Journal of Anhui Agricultural Sciences,2010,38(10):5002-5005. |
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| Authors: | YANG Kun |
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| Institution: | YANG Kun et al (College of Chemistry , Life Science,Zhejiang Normal University,Jinhua,Zhejiang 321004) |
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| Abstract: | Objective] The research aimed to isolate flanking sequences adjacent to the transgenic T-DNA in Brassica napus by an improved inverse PCR method.Method] Using single clone of transgenic FS4 in Brassica napus as the research materials,total DNA was extracted from transgenic Brassica napus by using modified CTAB method.After enzyme digestion and purification,self-joining was made.And two circles of nested PCR and the sequence alignment were made.Result] A fragement with the size of 4.0 kb was amplified aft... |
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| Keywords: | Inverse PCR (IPCR) Flanking sequences Improved CTAB method Transgenic Brassica napus |
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