RHO1基因的HIGS表达载体构建及转基因水稻的抗病性分析

Construction of HIGS expression vector of RHO1 gene and disease resistance of transgenic rice

【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。

[Objective] The host induced gene silencing （HIGS） expression vector of Magnaporthe oryzae RHO1 gene was constructed by Gateway technology,and then transformed into rice to study the re sistance to Magnaporthe oryzae. [Method] RH01 fragment was amplified by PCR from cDNA of Magna- porthe oryzae and pBDL03-RHO1 vector was constructed by Gateway technology before being transformed into Agrobacterium tumefaciens strain AGL1. Transgenic plants were generated by agrobacterium media- ted transformation, and then the spore of Magnaporthe oryzae was inoculated. The pathogenicity of Mag naporthe oryzae to transgenic rice was then analyzed. [Result] The RHC）I 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 Agrobacte- rium tumefaciens-mediated transformation, and the transgenic plants were verified by mCherry fluores- cence observation and PCR amplification. Further inoculation results showed that the transgenic rice had enhanced resistance to Magnaporthe oryzae infection. [Conclusion] Using Gateway technology can quicklyconstruct HIGS expression vector. Transgenic rice expressing RHOI-dsRNA had increased resistance to Magnaporthe oryzae ,indicating the role of RH01 mediated HIGS in rice-Magnaporthe oryzae interaction.