萼脊兰ACC氧化酶基因片段的克隆及其反义基因表达载体的构建

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.

Cloning and Construction of Antisense Plant Expression Vector of ACC Oxidase of Sedirea japonica

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.