摘要
以美洲商陆(Phytolacca americanaL.)为材料,利用RT-PCR方法克隆商陆抗病毒蛋白(PAP)的cDNA,并构建双元植物表达载体pBinARpap.用捕获该质粒的根癌农杆菌菌株LBA4404与烟草叶片外植体共培养,并在附加30 mg.L-1Kan的MS+0.1 mg.L-1IAA+1.5 mg.L-1BA的培养基上诱导植株分化,再生芽在附加30 mg.L-1Kan的MS培养基上生根,实现再生植株.Kan阳性植株经PCR-Sourthern检测筛选,得到PCR阳性植株,证明异源PAP基因在烟草基因组中的整合;经过RT-PCR检测,证明PAP基因在RNA转录水平上有表达;攻毒试验表明,转基因烟草植株对供试病毒TMV具有明显抗性.
Pokeweed antiviral protein gene of Phytolacca americana L. was one of the antiviral protein genes. Using the method of RT-RCR, it was cloned and inserted into binary vector under CaMV35 S promoter to construct the plant constitutive expression vector pBinARpap. The leave discs of tobacco were transformed with PAP gene via Agrobacterium-mediated transformation. Shoots were regenerated on MS medium supplemented with 30 mg·L^-1 Kan, 0.1 mg·L^-1 IAA and 0.5 mg·L^-1 BA and rooted on MS medium without hormone. PCR-Southern analysis proved PAP gene intergration in one Kanamycin resistance plantlet. RTPCR indicated the PAP gene of transgenic plantlet had expressed at the transcriptional level. Disease challenge test of the detached leaves of transgenic plantlet by inoculation of tobacco mosaic virus showed that the resistance was dramatically enhanced compared with non-transgenic plants.
出处
《河南农业大学学报》
CAS
CSCD
北大核心
2006年第2期130-132,141,共4页
Journal of Henan Agricultural University
