| 拟南芥热激蛋白HSP70基因片段克隆实验体系的建立 |
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| 引用本文： | 郭丽红,杨晓虹,刘开庆,袁燕.拟南芥热激蛋白HSP70基因片段克隆实验体系的建立[J].昆明师范高等专科学校学报,2009,31(6):46-48. |
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| 作者姓名： | 郭丽红 杨晓虹 刘开庆 袁燕 |
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| 作者单位： | 昆明学院生命科学与技术系,云南昆明650031 |
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| 基金项目： | 国家自然科学基金，云南省面上基金，云南省教育厅科学研究基金 |
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| 摘 要： | 以拟南芥为材料，对热激蛋白HSP70基因片段进行了克隆研究，获得了最佳的分子克隆实验体系．首先提取拟南芥总DNA，接着在热激蛋白HSP70编码区设计引物，扩增出HSP70编码区的片断，再将HSP70编码区的片断加上具有EcoRⅠ酶切位点双链的人工接头，通过EcoRⅠ对其进行酶切．再与经过EcoRⅠ酶切并已经去磷酸化的PUC19载体通过T4连接酶进行连接，然后将连接产物置人到感受态大肠杆菌细胞中（即转化），并让这种细胞在含有Amp＋／IPTG／X—Gal的LB培养基上生长．挑取培养基上蓝白斑中的白斑，进行质粒DNA提取，通过酶切质粒DNA，从而初步判断目的片段已被克隆．
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| 关 键 词： | 拟南芥 热激蛋白HSP70 基因片段 克隆 |
Establishment on Cloning Experimental System of Heat Shock Protein HSP70 in Arabidopsis thaliana |
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| GUO Li-hong,YANG Xiao-hong,LIU Kai-qing,YUAN Yan.Establishment on Cloning Experimental System of Heat Shock Protein HSP70 in Arabidopsis thaliana[J].Journal of Kunming Teachers College,2009,31(6):46-48. |
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| Authors: | GUO Li-hong YANG Xiao-hong LIU Kai-qing YUAN Yan |
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| Institution: | (Life Science and Technology Department, Kunming University, Yurman Kunming 650031, China) |
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| Abstract: | The seedlings of Arabidopsis thaliana were used as experimental material in the experiments. Firstly, Arabidopsis total DNA was obtained. Then, the coding region fragments of heat shock protein HSP70 were amplified by PCR with the primers which designed in the heat shock protein HSP70 coding region fragment. In order to connect with PUC19 vector, the gene fragments of heat shock protein HSP70 were linked with the double-stranded artificial joints With EcoR Ⅰ restriction site. After digestion with EcoR Ⅰ , the gene fragments of heat shock protein HSP70 were connected with PUC19 by T4 ligase, and then connected product were transformed into E. coli and grown in LB growth medium containing X-gal, IPTG and AMP. The white colonies were selected for plasmid DNA extraction and digested with EcoR Ⅰ. The result showed that gene fragments of heat shock protein HSP70 have been cloned. The optimal system for molecular cloning experiments was established. |
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| Keywords: | Arabidopsis thaliana Heat shock protein HSP70 gene fragments cloning |
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