摘要：

RPP13是一种能够通过识别病原物效应子诱发植物免疫反应的典型R基因。植物病毒学实验室前期对马铃薯纺锤块茎类病毒(PSTVd)所侵染的5个马铃薯品种进行了转录组测序分析,发现RPP13基因在5个马铃薯品种中均上调。为了研究该基因在马铃薯中是否与PSTVd抗性相关,应用DNAMAN 8.0软件对转录组测序所得到的RPP13上调基因序列进行序列比对分析,选取其共同的保守区域作为沉默对象,构建了以该保守区域为臂,马铃薯体细胞胚发生激酶类似受体基因(SERK1)(登录号为EF175216)内含子为环的茎环结构反向重复序列RNAi载体pROKⅡ-RPP13,通过冻融法将pROKⅡ-RPP13载体转入农杆菌LBA4404中,应用农杆菌介导的马铃薯遗传转化方法转化马铃薯品种民丰红,以苗龄28 d的健康马铃薯叶片为材料,经过预培养2 d、农杆菌浸染10 min、黑暗条件下共培养2 d,应用抗性筛选培养基诱导生根、生芽,将再生植株进行PCR检测,筛选得到3株阳性转基因植株;以马铃薯的ef1a基因作为内参基因,利用实时荧光定量PCR技术检测马铃薯转化植株中RPP13基因的表达量。结果表明:所获得的3株转基因植株中RPP13基因的表达量与未转基因的对照相比具有显著差异,在所获得的RNAi转基因马铃薯植株中,RPP13基因表达量的降低幅度为35%~60%。

关键词: 马铃薯, 遗传转化, RNAi, RPP13基因, 荧光定量PCR

Abstract:

RPP13 is a typical R gene that can induce plant immune response by recognizing of the effectors.The transcriptome sequencing analysis of five potato cultivars infected with Potato spindle tuber viroid (PSTVd)showed that RPP13 genes were up-regulated in the five potato cultivars infected with PSTVd.In order to study whether this gene was related to PSTVd resistance in potato,DNAMAN 8.0 software was used to perform sequence alignment analysis on RPP13 upregulated gene sequences obtained by transcriptome sequencing.The common conserved region was selected as the silencing object,and a stem-loop structure inverted repeat with the conserved region as the arm and the intron from somatic embryogenesis receptor-like kinase 1 (SERK1)(Accession number EF175216)as the loop was inserted in plant expression vector pROKⅡ to construct RNAi vector named pROKⅡ-RPP13.The pROKⅡ-RPP13 vector was transferred into Agrobacterium tumefaciens LBA4404 by freeze-thaw method,and the potato cultivar Minfenghong was transformed by Agrobacterium tumefaciens mediated genetic transformation method. Healthy potato leaves of 28 days of seedling age were used as materials. After 2 days of pre-culture, 10 minutes of infection, 2 days of co-culture under dark conditions, roots and buds were induced by the resistance screening medium. The regenerated plants were screened by PCR, and three positive transgenic plants were obtained.Using efla gene of potato as the reference gene,Real-time RT-PCR was used to detect the expression of RPP13 gene in potato transformed plants.The results showed that the expression level of RPP13 gene in the three transgenic plants obtained was significantly different from that in the control without transgenic.In the obtained RNAi transgenic potato plants,the expression level of RPP13 gene was decreased by 35%—60%.

Key words: Potato, Genetic transformation, RNAi, RPP13 gene, Fluorescence quantitative PCR