半嵌套式PCR检测转几丁质酶基因烟草研究

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Hind Ⅲ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。 

Study on the semi-nested PCR for the detection of tobacco expressing a chitinase gene

A sensitive method for detecting chitinase gene expressing tobacco plants and cured tobacco leaves is described. Medium containing Kanamycin was used to screen the transgenic tobacco seeds and seedlings. Then using western blot technique to further verify the presence of chitinase gene in the experimental samples. We studied the method of semi-nested PCR for testing transgenic tobacco expressing a chitinase gene. Tobacco leaf samples taken from transgenic tobacco plants were cured under certain high temperatures for about one week, and then tested for the presence of the chitinase gene also by semi-nested PCR. PCR products were further analyzed by digestion with Hind Ⅲ restriction endonuclease to verify the presence of the chitinase gene. Results showed that semi-nested PCR is a sensitive and rapid method for detecting chitinase expressing tobacco plants and cured tobacco leaved.