应用玉米核糖体失活蛋白基因改善马铃薯对立枯丝核菌抗性

Ribosome Inactivating Protein of Maize Enhanced Resistance to Rhizoctonia solani in Transgenic Potato

为探索在马铃薯中表达玉米源核糖体失活蛋白基因(RIP)是否可以改善受体对真菌病原立枯丝核菌(Rhizoctonia solani)的抗性,本研究以根癌农杆菌菌株LBA4404(p2301-RIP)转化马铃薯易感品种"Favorita"并进行了目的基因表达的分子生物学鉴定和对R.Solani的抗病性鉴定。结果表明:卡那霉素抗性再生株系的基因组中扩增到了目的基因,经Southern印记分析证实各独立转化株系基因组中均整合了2个拷贝RIP基因;RT-PCR分析表明RIP基因在转录水平正常表达;酶促活性测定表明部分转基因植株蛋白提取物具有RIP基因产物应有的脱嘌呤活性;温室盆栽试验证实有1个转基因株系显著提高了对由R.Solani引起的马铃薯黑痣病的抗性。

In the present study, the potato cultivar ‘Favorita＇was transformed via Agrobacterium tumefaciens strain LBA4404 containing the plasmid p2301-RIP which harbors the Ribosome Inactivating Protein（RIP）. The potato leaf discs were used as an explant for transformation. The PCR technique was used for identification of transformed plants. Southern blot and RT-PCR analyses were applied for molecular characterization of the transgenic clones, and the depurination activity of ribosome were detected for RIP protein of the transgenic clones.A greenhouse assay was carried out to evaluate the resistance to Rhizoctonia solani pathogen of transgenic clones expressing the RIP gene. The results revealed that part of the plants developed in selective medium were positive for the corresponding gene using the PCR technique. Southern blot analysis demonstrated that the tested transgenic plants integrated two copies of RIP gene into their genome. The expression of the RIP protein was confirmed in the leaf extracts of the transgenic clones by depurination activity analysis of ribosome. Resistance evaluation of the transgenic plants in greenhouse conditions showed that disease severity were reduced for R. solani.