蝴蝶兰ACC氧化酶和ACC合成酶融合反义表达载体的构建

Construction of Double-antisense Expression Vector of ACC Oxidase and ACC Synthase Gene in Phalaenopsis

为了获得具有超常花期的蝴蝶兰新品系,根据已报道的蝴蝶兰ACC合成酶(ACS)基因和ACC氧化酶(ACO)基因的序列,设计引物从蝴蝶兰总RNA中克隆出ACS保守区的461 bp片段和ACO的333 bp片段,完成2个基因的克隆和测序后,将ACS基因片段和ACO基因片段以反义的方向插入到中间载体pBI221 CaMV启动子的下游,酶切后获得含有CaMV启动子的ACS和ACO基因的反义融合片段。将此包含启动子的反义融合片段连接到植物表达载体pCAM-BIA3301中,获得ACC合成酶基因和ACC氧化酶基因融合的反义表达载体,将构建好的植物表达载体转化农杆菌LBA4404,用于侵染蝴蝶兰原球茎。

According to the reported ACC oxidase （1-aminocyclopropane-1-carboxylate oxidase, ACO） gene and ACC synthase（ACS） gene cDNA sequences,two pairs of specific primers were designed and synthesized,which were obtained from total RNA of Phalaenopsis by RT-PCR. The cDNA fragments were linked into a plant binary vector pBI221 between the CaMV 35S promoter and nos terminator. Then the recombinant fragment including ACC, ACO and 35S promoter was cloned into plant expression vector pCAMBIA3301 in orientation. The positive clones were detected by PCR technique and analyzed by the restriction enzyme. The constructs were transferred into agrobacterium LBA4404 via frozen-fusion method and had been used to transform Phalaenopsis protocorrn by agrobacteria.