蝴蝶兰ACC氧化酶cDNA片段的克隆及其反义植物表达载体的构建

cDNA Cloning of ACC Oxidase Gene of Phalaenopsis and Construction of Its Antisense Plant Expression Vector

根据已报道的蝴蝶兰ACC氧化酶的基因序列,设计了一对引物,通过RT-PCR从蝴蝶兰总RNA中扩增出ACC氧化酶的cDNA片段,将其连接到pMD19-T载体上进行测序.结果显示,该片段与参考的已报道序列具有99.70%的同源性.测序后将该基因的cDNA序列反向插入pBI221的CaMV35S启动子和nos terminator之间,构建了蝴蝶兰ACC氧化酶的反义基因植物表达载体,为进一步转化蝴蝶兰研究其对蝴蝶兰花期和生长的影响打下了基础.

A pair of specific primers were designed and synthesized according to the reported cDNA sequences of the ACC oxidase （1-aminocyclopropane-l-carboxylate oxidase, ACO）gene of Phalaenopsis. The partial eDNA of ACO was obtained from total RNA of Phalaenopsis by RT-PCR. Then the eDNA fragment was linked to T-tailing pMD19-T vector for cloning and sequencing. The results show that the obtained sequence was made up of 333 bp and the homologous rate was 99.70 % compared with the reported sequences. The antisense expression vector was constructed by inserting the fragment of ACO into pBI221 between the CaMV35S promoter and nos terminator in antisense orientation. The work laid foundation for further study on the transformation of Phalaenopsis with antisense ACO gene.