| 摘要: |
| 【目的】使用Gateway技术构建稻瘟菌RHO1基因寄主诱导的基因沉默（HIGS）表达载体，将其转化入水稻后，观察稻瘟菌对转基因水稻的致病力。【方法】利用PCR技术从稻瘟菌cDNA扩增RHO1基因，通过Gateway技术构建pBDL03-RHO1载体，电转化进入农杆菌AGL1；以水稻日本晴为材料，通过农杆菌介导的转化方法获得转基因水稻植株，并对其接种稻瘟菌，分析转基因水稻对稻瘟菌抗性。【结果】通过 PCR 扩增获得了RHO1基因片段，利用BP反应构建了入门载体 pDONR 221-RHO1，采用LR反应构建了HIGS表达载体pBDL03-RHO1。通过农杆菌介导的转基因方法将HIGS载体转入了水稻中，并利用mCherry红色荧光观察和 PCR 扩增验证了转基因植株构建成功。接种试验结果表明，HIGS转基因植株对稻瘟菌的抗性提高。【结论】利用Gateway技术可以简单快速构建HIGS表达载体，表达RHO1的转基因水稻抗病性提高，表明RHO1介导的 HIGS 在水稻-稻瘟菌互作过程中可以发挥作用。 |
| 关键词: 稻瘟菌 寄主诱导的基因沉默 水稻 抗病性 Gateway技术 |
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| 基金项目:国家自然科学基金青年项目（31301607）；国家重点基础研究发展计划（“973”计划）项目（2013CB127700） |
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| Construction of HIGS expression vector of RHO1 gene and disease resistance of transgenic rice |
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TANG Zhe,ZHANG Qiang,XIANG Ping
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| Abstract: |
| 【Objective】The host induced gene silencing (HIGS) expression vector of Magnaporthe oryzaeRHO1 gene was constructed by Gateway technology,and then transformed into rice to study the resistance to Magnaporthe oryzae.【Method】RHO1 fragment was amplified by PCR from cDNA of Magnaporthe oryzae and pBDL03-RHO1 vector was constructed by Gateway technology before being transformed into Agrobacterium tumefaciens strain AGL1.Transgenic plants were generated by agrobacterium mediated transformation,and then the spore of Magnaporthe oryzae was inoculated.The pathogenicity of Magnaporthe oryzae to transgenic rice was then analyzed.【Result】The RHO1 gene fragment was amplified by PCR,and the entry vector pDONR 221-RHO1 was constructed by BP reaction.The HIGS expression vector pBDL03-RHO1 was constructed by LR reaction.The HIGS vector was transferred into rice by Agrobacterium tumefaciens mediated transformation,and the transgenic plants were verified by mCherry fluorescence observation and PCR amplification.Further inoculation results showed that the transgenic rice had enhanced resistance to Magnaporthe oryzae infection.【Conclusion】Using Gateway technology can quickly construct HIGS expression vector.Transgenic rice expressing RHO1 dsRNA had increased resistance to Magnaporthe oryzae,indicating the role of RHO1 mediated HIGS in rice Magnaporthe oryzae interaction. |
| Key words: Magnaporthe oryzae HIGS rice disease resistance Gateway technology |
