烟草马铃薯Y病毒病相关基因eIF4E的片段克隆及RNAi载体构建

Cloning of PVY Associated Gene eIF4E and Construction of Its Corresponding RNAi Vectors

以在植物体内转录出RNA沉默的高效诱导因子双链RNA(dsRNA)为目标,以感病烟草品种为材料,提取总RNA,根据已报道的eIF4E基因的核苷酸序列,在保守区域和差异区域分别设计特异性引物,采用RT-PCR克隆出eIF4E基因的目的片段。将正向目的基因用XbaⅠ和ClaⅠ切下,将所得基因片段与用同样酶切开的含内含子的载体pKAN相连,取名pK-S;用同样的方法,将反向目的基因用XhoⅠ和KpnⅠ切下,所得基因片段与用同样酶切开的pK-S相连,取名pK-SA,再用NotⅠ将包括Intron和正反向基因在内的片段切下,将其定向插入同样酶切开的pART27上,获得重组质粒。以中间载体pKANNIBAL和表达载体pART27为基础,构建成以CaMV35S启动子为驱动的含有"正向eIF4E目的片段-pdkintron-反向eIF4E目的片段"的RNAi烟草表达载体,获得的载体可以应用于植物转基因抗病毒育种工程中。

In order to effectively transcribe dsRNA in plants,an inducer of RNA silencing,the total RNA was isolated from the susceptible tobacco variety,the specific primers of the high homology and differential region was designed according to the published sequence of eIF4E gene,cloned the target gene fragment of eIF4E gene by RT-PCR.Sense fragment was obtained by digesting with XbaⅠ and ClaⅠ and inserted into vector pKAN,then called the recombinant plasmid pK-S.Antisence gene cut with XhoⅠ and KpnⅠ was linked with pK-S to construct hairpin structure.The fragment including intron,the sense and antisence gene was cut by Not Ⅰ,and was inserted into pART27 to construct plant transformation vector.Then the promoter CaMV 35S driven,containing Sense eIF4E gene target fragments-pdk intron-Antisence eIF4E gene target fragments,tobacco RNAi expression vector were constructed based on the intermediate vector pKANNIBAL and expression vector pART27.The vector could be applied in transgenic breeding engineering for plant viral resistance.