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用含卡那霉素的培养基首先对转几丁质酶基因烟草种子和烟苗进行初步筛选鉴定 ,再用 Western Blot进一步证实抗卡那霉素的转基因烟苗中外源转几丁质酶基因的表达。然后研究半嵌套式 PCR(Semi- nested PCR)检测转几丁质酶基因烟草植株及其烤后烟叶中的所转目的基因 ;利用限制性内切酶 H ind 对半嵌套式 PCR产物进行酶切 ,从而验证半嵌套式 PCR产物是否为真正的目的基因。研究结果表明 ,半嵌套式 PCR是一种快速、灵敏的检测转几丁质酶基因烟草植株及烤后烟叶的方法
The kanamycin-containing medium was firstly used to screen and identify tobacco seeds and tobacco seedlings which were transferred to chitinase gene. Western Blot was used to further confirm that the exogenous chitinase gene expression. Then semi-nested PCR (semi-nested PCR) was applied to detect the target gene in tobacco plants transformed with chitinase gene and its roasted tobacco leaves. The semi-nested PCR products were digested with restriction endonuclease H ind Digested to verify whether the semi-nested PCR product is the real target gene. The results show that semi-nested PCR is a rapid and sensitive method for detecting tobacco plants transformed with chitinase gene and cured tobacco leaves