同源重组快速构建百合LCHS2基因RNAi表达载体及其鉴定

Rapid Construction and Identification of RNAi Expression Vector of LCHS2 Gene from Lilium Based on the Principle of Homologous Recombination

为探讨CHS基因的下调表达对花朵着色的影响以及为百合观赏性状遗传改良提供技术支持,利用基于同源重组原理的Gateway技术,根据课题组从东方百合‘索邦’分离的LCHS2基因序列(Genbank登录号:GQ483376)和pENTR/D-TOPO载体要求,设计上游5'端添加'CACC'的一对特异引物,PCR扩增获得636 bp的干扰片段。通过TOPO克隆,将该片段克隆入载体pENTR/D-TOPO构建入门克隆载体pENTR/D-CHS,测序结果显示,与原序列同源性为100%。再经过LR反应使pENTR/D-CHS上的干扰片段替换掉目标载体pHellsgate12上的ccdB片段,快速构建了包含LCHS2基因干扰片段的RNAi表达载体pH12-CHSi,经限制性内切酶XhoI、XbaI酶切鉴定获得724 bp和722 bp的片段,与预期结果一致。pH12-CHSi电击法转化农杆菌GV3101,菌液PCR鉴定,出现636 bp的目的条带,表明RNAi表达载体pH12-CHSi已成功转入农杆菌。

In order to discuss the effect of down-regulated expression of CHS gene on pigmentation of flowers and to offer technological support for genetic modification of ornamental characteristics in lilium. The Gateway technology based on homologous recombination was adopted and per the requirements of pENTR/D-TOPO vector and LCHS2 gene sequence （Genbank accession NO： GQ483376） from Oriental Lily cv ＂Sorbonne＇, a special pair of primers were designed, the upstream of which beared ＂CACC＂ at 5＂ end. An interference fragment of 636bp was obtained by PCR amplification with the primers above and cloned into pENTR/D-TOPO to form pENTR/D-CHS via TOPO cloning. The sequencing result of pENTR/D-CHS showed that the interference fragment was 100% identical to LCHS2 gene. Then the ccdB fragment on destination vector pHellsgatel2 was replaced by the interference fragment on pENTR/D-CHS via LR reaction to form RNAi expression vector pH12-CHSi, which was digested by restriction enzyme XhoI and XbaI and the expected fragments of 724 bp and 722 bp appeared, pH12-CHSi was transformed into Agrobacterium strain GV3101 by electroporation and the expected fragments of 636 bp appeared in the performance of bacteria liquid PCR.