虎杖PcPKS1基因RNAi载体的构建

Construction of RNAi Vector Targeting Pc PKS1 Gene of Polygonum cuspidatum Sieb.et Zucc.

虎杖(Polygonum cuspidatum Sieb.et Zucc.)是富含芳香族聚酮化合物的药用植物,Ⅲ型聚酮合酶(Polyketide synthase,PKS)对芳香族聚酮化合物的合成起重要作用。为了研究虎杖Ⅲ型聚酮合酶基因Pc PKS1的功能,分别选取Pc PKS1基因的编码区保守序列(574 bp)和3′端非编码区特异序列(209 bp)作为干扰片段。将选定的干扰片段,分别以正向和反向插入到p YLRNAi载体中GUS基因内含子的两侧,以形成hp RNA表达盒,成功构建了以组成型Ca MV35S为启动子,以潮霉素抗性为筛选标记的RNA干扰表达载体p YLRNAi-CDS和p YLRNAi-UTR,并将载体导入农杆菌LBA4404中。

Polygonum cuspidatum Sieb.et Zucc. is a medicinal plant rich in aromatic polyketides. Type III polyketide syn- thases （PKSs） are thought to play a crucial role for construction of the basic skeleton of a variety of the aromatic polyke- tides. In order to conform the function of the type III PKS gene PcPKS1 from Polygonum cuspidatum Sieb.et Zucc.,two dif- ferent fragments targeting the conservative coding region （574 bp in length） and the 3＇-untranslated region （209 bp in length） of PcPKS1 were selected to construct RNA interference （RNAi） expression vector,respectively. These fragments were inserted into pYLRNAi flanking GUS intron in sense orientation and antisense orientation,thus forming hpRNA cassettes. The vectors, namely,pYLRNAi-CDS and pYLRNAi-UTR,were composed of inversely-repeated sequence fragments,an constitutive promoter CaMV35S and a hygromycin resistance gene as a selectable marker.Finally the vectors were introduced into A- grobacterium LBA4404.