大花蕙兰转齿兰环斑病毒外壳蛋白基因及检测

Genetic Transformation and Detection of the Cymbidium hybridum Modified by Coat Protein Gene of ORSV

[目的]通过植物转基因技术获得抗病毒大花蕙兰种质资源,优化转化体系和鉴定方法。[方法]本研究克隆了齿兰环斑病毒外壳蛋白基因,并构建了该基因的p BI121表达载体,用根癌农杆菌介导法转化大花蕙兰,尝试以巢式PCR方法检测转基因再生植株。[结果]优化了大花蕙兰遗传转化体系,建立了利用巢式PCR技术检测转基因大花蕙兰植株的方法,获得了32株转基因株(系)。[结论]优化了以类原球茎为外植体的农杆菌介导转化大花蕙兰的方法,确定以5%10%类原球茎存活时的抗生素(卡那霉素)浓度为筛选浓度;获得了转ORSV CP基因大花蕙兰植株;对大花蕙兰转基因植株检测时,巢式PCR较普通PCR更灵敏、准确。

[Objective]To optimize the transformation system and identification methods for obtaining the germplasm resources of anti-virus Cymbidium hybridum by plant transgenic technology. [Method]The coat protein gene（ CP）of ORSV was cloned from the c DNA of infected C. hybridum. After sequencing,the gene was constructed into p BI121 expression vector and transformed to the protocorm-like bodies（ PLBs） of C. hybridum by Agrobacterium tumefaciens-mediated method. [Result]The genetic transformation system was optimized and the identification method of the transgenic plants was established by nest polymerase-chain-reaction（ Nest-PCR）. There were 32 transgenic plants of C. hybridum detected by Nest-PCR. [Conclusion]The Agrobacterium tumefaciens-mediated transformation method of C. hybridum was optimized using PLBs as explants. The antibiotics（ kanamycin） screening concentration was determined as 5% to 10% PLBs survival rate. Using the Nest-PCR to detected the transgenic plants was more sensitive and accurate than conventional PCR.