NtSKP1基因的RNAi载体构建及对烟草的转化

Construction of RNAi Expression Vector of NtSKP1 and Transformation in Tobacco

根据枯斑三生烟草SKP1基因(NtSKP1)的序列,设计二对分别含有特定酶切位点的特异引物,以重组质粒pMD18-SKP1为模板,分别扩增目的基因的正反向片段(约473 bp)。将反向目的片段插入中间载体pHANNIBAL内含子右侧,正向目的片段插入该载体内含子左侧,再经NotⅠ酶切回收约3 916 bp目的片段,插入到双元载体质粒pART27中,成功构建了含NtSKP1基因片段正反向重复序列的植物表达载体pART27-skp1sa。将pART27-skp1sa质粒导人根癌农杆菌LBA4404中并转化烟草叶片细胞,经选择分化培养,获得转基因烟草。

According to the sequence of SKP1 from Nicotiana tabacum var.Samsun NN（NtSKP1）,two pairs of specific primers containing special restriction enzyme sites were designed.The target reverse and forward fragments（473 bp）,were amplified and inserted into right and left side of intron in intermediated vector pHANNIBAL respectively.Then the target fragment about 3916 bp was cut by NotⅠ from recombinant intermediated vector,and cloned into binary plasmid pART27.Plant expression plasmid pART27-skp1sa,with inverted repeat DNA fragment of NtSKP1,was successfully constructed,and pART27-skp1sa was introduced into Agrobacterium tumefaciens LBA4404.Then transformed tobacco leaves,and the transgenic plants of tobacco were obtained.