| 摘要: |
| 【目的】为方便基于植物瞬时表达技术的高通量功能基因筛选,利用Gateway技术实现植物表达载体和表达文库的构建，将35S启动子和Gateway技术入门载体pDONR222的同源重组区连接到植物表达载体pCAMBIA0380上，构建Gateway技术兼容的植物表达载体。【方法】分别扩增35S启动子与pDONR222上attP特异识别序列之间的功能区，并将其依次连入根癌农杆菌(Agrobacterium tumefaciens)介导的植物表达载体pCAMBIA0380的多克隆位点区，利用带有attB特异识别区域的GUS基因，对构建的载体功能进行测试。【结果】成功构建了基于Gateway技术的植物表达载体p1104D；载体重组基因的选择性测试结果表明，p1104D对目的基因片段的大小无严格选择性；载体重组效率测试结果表明，p1104D重组平均滴度为5.11×105 cfu/mL；GUS报告基因瞬间表达试验结果表明，改造后的载体可以实现目标基因的顺利表达。【结论】基于Gateway技术的植物表达载体p1104D的构建，为实现cDNA文库的高效构建和目标基因的高通量功能筛选提供了可能，有望推动植物 病原互作研究中关键基因的鉴定与克隆。 |
| 关键词: Gateway技术 植物表达载体 瞬时表达 功能克隆 |
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| 基金项目:科技部“973”计划项目（2006CB101901）；高等学校学科创新引智计划项目（B07049） |
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| Construction of a Gateway technology-based plant expression vector |
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| Abstract: |
| 【Objective】To facilitate plant gene expression research and transient expression based high throughput functional gene cloning in plants,the 35S promoter sequence from pBi121 and the recombination specific sites attP spanning region from pDONR222 were inserted into pCAMBIA0380 to make Gateway technology-compatible Agrobacterium tumefaciens mediated plant expression vector.【Method】The 35S promoter sequence and the attP spanning region in the Gateway vector pDONR222 were amplified and inserted into the multiple cloning sites of A.tumefaciens mediated plant expression vector pCAMBIA0380 to make p1104D.The functionality of the constructed vector p1104D was tested by cloning multiple genes in the same BP reaction and by GUS gene expression.【Result】A Gateway technology-compatible A.tumefaciens mediated plant expression vector named p1104D was constructed.The average titer of p1104D was 5.11×105 cfu/mL and it showed no preference for different size fragments of DNA in BP reaction.Its functionality was confirmed by successful detection of GUS activity in onion epidermal cells by A.tumefaciens mediated transient expression.【Conclusion】The constructed Gateway technology-compatible A.tumefaciens mediated plant expression vector makes it possible to combine cDNA library construction and high throughput functional gene cloning,and is expected to facilitate identification and cloning of genes that play important roles in plant pathogen interaction. |
| Key words: Gateway technology plant expression vector transient expression functional cloning |
