植物介导的RNA干扰引起马铃薯晚疫病菌基因的沉默

Gene Silence of Phytophthora infestans Induced by Plant-mediated RNA Interference in Potato

由致病疫霉(Phytophthora infestans)引起的晚疫病是最具毁灭性的马铃薯病害。为明确植物介导的RNAi沉默致病疫霉基因的有效性,本研究采用重叠延伸PCR技术克隆同时与晚疫病菌4个ces基因均同源的融合基因C1234,构建内含子连接的C1234反向重复序列植物表达载体,采用农杆菌介导法转化晚疫病易感马铃薯品种大西洋,经PCR和Southern杂交检测,获得129个转基因株系。离体叶片接种病原菌后,有97个转基因株系发病速度明显慢于野生型,接种6 d后病斑大小和霉层厚度均明显小于对照,并且叶片感病部位没有出现失绿斑,而野生型产生了明显的失绿斑。实时定量RT-PCR分析发现,发病延缓的叶片上致病疫霉4个纤维素合酶基因的表达水平明显低于野生型。本研究表明,转基因植株中产生的以晚疫病菌ces基因为靶标的ds RNA能够沉默致病疫霉相应基因表达,延缓发病进程。

Potato late blightcaused by Phytophthora infestans is the most devastating disease in potato. The objective of this study was to test the efficiency of plant-meidated RNAi in silencing genes in P. infestans and to find a new way to breed potato resistant to late blight. Over-lap PCR was employed to amplify a fused-geneC1234 simultaneously homologous to four cellulose synthase genes inP. infenstans. Then, a plant expression vector containing inverted repeat ofC1234was constructed and transferred to Atlantic, a potato variety severely susceptible to late blight by agrobacteria-mediated genetic transformation. A hundred and twenty nine regenerated lines were confirmed to be transgenic plants with PCR and Southern blot. When detached leaves were inoculated withP. infestans, 97 out of 129 transgenic lines delayed disease symptoms with smaller lesions and less hyphae com-pared to the wild type at six days after inoculation. Chlorotic spots did not appeared on leaves from transgenic lines while deve-loped severely on leaves from the wild type at the same day. mRNA accumulation of the four cellulose synthase genes fromP. infestanscolonized on leaves from transgenic plants with delayed symptoms was significantly lower compared to the wild type with Real-time RT-PCR.The results demonstrated that dsRNA ofC1234 generated in transgenic potato could induce homologous genes silence and delay the process of infection in intimately touchedP. infestans.