植物免疫调控基因RTP5(Resistance to Phytophthora 5)编码一个含WD40结构域的蛋白,该基因负调控植物对疫霉的抗性.为获得沉默马铃薯中RTP5同源基因StRTP5a和StRTP5b的遗传材料,以便进一步探究StRTP5a和StRTP5b基因的生物学功能.以四倍体马铃薯品种'Desiree'的cDNA为模板,扩增到靶向StRTP5a和StRTP5b基因的DNA片段,通过Gateway技术将目的片段重组至植物表达载体pHells-gate12,构建同步靶向StRTP5a和StRTP5b基因的目的表达载体pHells12-StRTP5a/b i,然后利用根癌农杆菌介导的遗传转化方法将目的表达载体转化至'Désirée'中.经PCR和qPCR分析后,获得8株2个目的基因同时沉默的马铃薯株系,转化株系中StRTP5a和StRTP5b基因表达量同时降幅最高达70％以上.
Construction of an RNAi Vector Targeting both StRTP5a and StRTP5b Genes in Potato and Generation of Transgenic Potato Lines with Silenced StRTP5a and StRTP5b
The WD40 protein-encoding geneRTP5(Resistance to Phytophthora 5)acts as a novel reg-ulator of plant immunity,negatively regulating plant resistance to Phytophthora pathogens.This study aimed to obtain transgenic potato lines with concurrent silencing of both StRTP5a and StRTP5b genes for further functional analysis.A DNA fragment targeting both StRTP5a and StRTP5b genes was amplified from the cDNA of the tetraploid potato cultivar'Désirée'and inserted into the plant ex-pression vector pHellsgate12 using the gateway recombination cloning technology.The resultant desti-nation vector,pHells12-StRTPa/b.targeting both StRTP5a and StRTP5b genes,was successfully generated.Subsequently,it was introduced into potato cultivar'Désirée'via Agrobacterium tumefa-ciens-mediated genetic transformation.Eight transgenic potato lines with simultaneous silencing of both target genes were obtained after thorough PCR and qPCR analysis.Expression levels of both StRTP5a and StRTP5b genes were reduced by up to 70％or more in transgenic lines compared to the wild-type potato cultivar'Désirée'.
PotatoRNAiGenetic transformationWD40 domain-containing protein
万方数据
维普
