CAJ | 학술논문

根据已报道的几种不同属的兰科植物ACO基因序列,设计并合成了一对特异引物,以萼脊兰的基因组DNA为模板,用PCR扩增方法克隆出萼脊兰ACO基因的部分片段,将其连接到pMD19-T质粒载体上进行测序.结果显示,该片段的长度为333 bp,序列和蝴蝶兰(Phalaenopsis hybrid)、卡特兰(Cattleya intermedia)、两棱蕾丽兰(Laelia anceps)的相应ACO片段的同源性分别达到达到99.7%、92.49%和92.19%;和大花蕙兰(Cymbidium hybrid)、石斛兰(Dendrobium crumenatum)的同源性分别是89.49%和89.19%.将此片段反向插入植物表达载体pBI221的CaMV35S启动子和NOS终止子之间,成功构建了萼脊兰ACO的反义基因植物表达载体pBI-antiACO.
근거이보도적궤충불동속적란과식물ACO기인서렬,설계병합성료일대특이인물,이악척란적기인조DNA위모판,용PCR확증방법극륭출악척란ACO기인적부분편단,장기련접도pMD19-T질립재체상진행측서.결과현시,해편단적장도위333 bp,서렬화호접란(Phalaenopsis hybrid)、잡특란(Cattleya intermedia)、량릉뢰려란(Laelia anceps)적상응ACO편단적동원성분별체도체도99.7%、92.49%화92.19%;화대화혜란(Cymbidium hybrid)、석곡란(Dendrobium crumenatum)적동원성분별시89.49%화89.19%.장차편단반향삽입식물표체재체pBI221적CaMV35S계동자화NOS종지자지간,성공구건료악척란ACO적반의기인식물표체재체pBI-antiACO.
A pair of specific primers were designed and synthesized according to the reported DNA sequences of the ACC oxidase gene of Phalanopsis,Cattleya,Laelia,Cymbidium,Dendrobium.The partial DNA of ACO was obtained from genomic DNA of Sedirea japonica by PCR.Then the DNA fragment was linked to T-tailing pMD19-T vector for cloning and sequencing.The results showed that the obtained sequence was made up of 333 bp and the homologous rates were 99.7%,92.49% ,92.19%,89.49% and 89.19% compared with the relevant fragments of reported ACO sequences of Phalaenopsis hybrid,Cattleya intermedia,Laelia anceps ,Cymbidium hybrid and Dendrobium crumenatum.The antisense expression vector was constructed by inserting the fragment of ACO into pBI221 between the CaMV35S promoter and NOS terminator in antisense orientation.