RNAi介导的百合抗CMV和LMoV的载体构建

为获得高效的RNAi(RNA interference)表达载体，培育百合抗病毒新种质，选取感病百合叶片为试验材料，同源序列比对后选取CMV 2b基因271 bp和LMoV cp基因428 bp作为靶基因片段。首先采用RT-PCR方法对感病植株进行病毒检测，确定其带有CMV和LMoV病毒；之后提取感病植株叶片总RNA，反转录得到cDNA，设计带有不同酶切位点的引物，以反转录得到的cDNA为模板，PCR扩增获得目的片段，产物回收纯化后，转化大肠杆菌，重组克隆。将各自的反向片段、正向片段顺次连接到载体pFGC5941上，经多次双酶切、连接、转化后，采用冻融法将构建好的载体转入农杆菌EHA105并进行重组鉴定。酶切结果表明已成功构建了分别抗CMV、LMoV的RNAi植物表达载体pFGC5941-C2和pFGC5941-L2，PCR扩增结果表明表达载体已成功转入农杆菌中。所构建的载体及获得的工程菌与预期的完全一致，获得含载体的农杆菌可以直接应用于百合转基因抗病毒育种工程中。

Construction of RNAi Expression Vectors Respectively Resistant to CMV and LMoV

In order to obtain the efficient expression vectors for new lily antiviral germplasm, CMV 2b gene 271 bp and LMoV cp gene 428 bp fragments were chosen as the target sequence to construct the vectors by homologous sequence. The required virus was confirmed by RT-PCR method from infected leaves. And then PCR method was used to obtain the cDNA from the total RNA. Using the cDNA as template, the target fragments were obtained under specific primers with different restriction enzyme cutting sites. The two fragments were inserted into both sides of intron of pFGC5941 in forwards and reverse after recombinant clone. The RNAi vector was introduced into Agrobacterium tumefaciens EHA105. Restriction enzyme digestion showed that the RNAi vectors were constructed successfully. The results of PCR confirmed that both of the two vectors pFGC5941-C2 and pFGC5941-L2 were introduced into Agrobacterium tumefaciens EHA105. The results of PCR and sequencing showed that the constructed vectors and the engineering bacteria were completely consistent with the expected, the RNAi vectors were constructed successfully, which could be applied in transgenic breeding engineering for lily viral resistance.