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病毒病是制约马铃薯生产的重要因素之一。在拟南芥等植物的研究中发现,抑制寄主因子可以显著降低细胞中病毒的累积量,从而缓解病害症状。本研究在获得与拟南芥寄主因子AtTOM1和AtTOM3具有同源性的马铃薯基因StTOM1和StTOM3的基础上,尝试用RNAi方法同时沉默StTOM1和StTOM3。以pUCCRNAi为中间载体,构建同时含StTOM1和StTOM3双基因干扰片段StT1-StT3的质粒pUCStT1-StT3-dRi(±),再将双基因干扰片段StT1-StT3切下并连接到双元载体pBI121上。用农杆菌介导方法,将StT1-StT3片段导入马铃薯中,获得转基因马铃薯小苗,阳性率达到83.6%。RT-PCR检测表明,转StT1-StT3马铃薯中StTOM1基因mRNA的表达水平下调了78%,StTOM3基因下调了81%。StTOM1和StTOM3沉默转基因马铃薯的获得,为将来验证和评价StTOM1和StTOM3是否为马铃薯病毒的寄主因子及在创建抗病毒马铃薯新种质的潜力,奠定了基础。
Virus disease is one of the important factors restricting potato production. In the study of Arabidopsis and other plants, it was found that the inhibition of the host factor can significantly reduce the cumulative amount of virus in the cells, thereby alleviating the symptoms of the disease. In this study, StTOM1 and StTOM3, which are homologous to Arabidopsis thaliana host genes AtTOM1 and AtTOM3, were stained with RNAi. Plasmid pUCStT1-StT3-dRi (±) containing both StTOM1 and StTOM3 double stranded plasmids StT1-StT3 was constructed by using pUCCRNAi as an intermediate vector. The double stranded plasmids StT1-StT3 were excised and ligated into binary vector pBI121. Using Agrobacterium-mediated method, StT1-StT3 fragment was introduced into potato to obtain transgenic potato seedlings, the positive rate reached 83.6%. RT-PCR results showed that StTOM1 mRNA expression in StT1-StT3 transgenic potato was down-regulated by 78% and StTOM3 gene by 81%. The availability of StTOM1 and StTOM3-silenced transgenic potatoes laid the foundation for the future validation and evaluation of whether StTOM1 and StTOM3 are host factors of potato virus and their potential to create new antiviral potato germplasms.