百合花发育相关基因LLGLO1的RNAi载体构建

Construction of RNAi vector of LLGLO1 gene related to flower development of Lilium

百合花发育相关基因的研究是开展花卉分子育种的重要基础。以麝香百合(Lilium longi-florumThumb.)未开放的花蕾为材料提取总RNA。根据LLGLO1序列的同源比对选取特异区段,设计引物扩增区段cDNA并引入酶切位点。将PCR扩增片断连接到T载体上,经酶切及PCR验证后进行测序。测序结果表明:用于RNAi的cDNA片断与LLGLO1序列完全相同。通过中间载体pSK+intron引入一段内含子间隔区,RNA干扰片断经过多次的酶切和连接过程最终连入表达载体pCAMBIA1301中形成ihpRNA结构。PCR及双酶切鉴定结果显示,构建的RNA干扰载体的ihpRNA结构完整。

Dissecting the genes that are related to flower development are essential for lily molecular breeding. The total RNA was isolated from the flower bud of Lilium longiflorum Thumb. According to the homologous alignment of LLGLO1, the specific cDNA segment was chosen, onto which the restriction site was inserted. The PCR amplification fragment was connected to the T-vectors and sequenced after restriction enzyme digestion and PCR. The sequencing results showed that the cDNA fragment used in RNAi and LLGLO1 were identical. RNA interference fragment was finally connected to the expression vector pCAMBIA1301 to form the ihpRNA structure through subcloning an intron spacer by intermediate vector pSK ＋ intron and the subsequent repeated processes of restriction enzyme digestions and connections. The result of PCR and double digestion verification showed that the structure of the ihpRNA of the RNA interference vector was intact.