花生抗黄曲霉的植物双价表达载体的构建

Construction of Plant Bivalent Expression Vectors Against Aflatoxin in Peanut

通过常规育种培育的抗黄曲霉品种皆表现抗性不稳定,鉴此开展通过基因工程手段以培育高抗以及无筛选标记的转基因花生品种。几丁质酶基因CHI和葡聚糖酶基因GLU皆为广谱性的抗病基因且具有协同增效作用。分别以pSC1300-8-CHI和pSC1300-13-GLU载体为基础,通过PCR技术在CHI基因的特异启动子8#前端和T-nos末端分别加入NcoⅠ酶切位点和AflⅡ酶切位点,在GLU基因的特异启动子13#前端和T-nos末端分别加入ApaⅠ酶切位点和SpeⅠ酶切位点,通过酶切、连接将上述2个基因连接在抗生素自我删除载体pLoxp上,获得了具有抗生素自我删除选择标记的双价果种皮特异表达载体pLoxp-8-CHI-13-GLU。经过限制性内切酶鉴定,该植物表达载体构建成功。

The performance of species resistance to Aspergillus by conventional breeding are unstable, therefore, genetic engineering is used to breed varieties resistant to Aspergillus. LHI and GLU are broad-spectrum resistance genes and have synergistic effect. Based on constructed vectors pSC13OO-8-CHI and pSC13OO-13-GLU, Nco I restriction site was added to the front of the promotor 8# of CHIgene and AflII restriction site was added to the end of T-nos sequence through PCR amplification, Apa I restriction site was added to the front of the promotor 8# of GLU gene and Spe I restriction site was added to the end of T-nos sequence through PCR amplification, two genes will be connected to antibiotics serf-removing carrier pLoxp after digestion, antibiotic induced-removing plant bivalent expression vectors pLoxp-8-CHI-13-GLU was constructed. Identified by restriction endonuclease, the expression vector was successfully constructed.