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利用Act1启动子和除草剂选择标记bar基因,构建了含有小麦黄花叶病毒外壳蛋白基因的植物表达载体,并采用基因枪法导入小麦品系.经PCR和PCR-RFLP对转基因小麦T0代及T1代进行检测后,对阳性植株的后代株系(T2代)进行田间抗病性检测.结果表明,外源基因可以在后代中遗传,并且其中一个转基因株系的后代(P8-T2)对小麦黄花叶病毒呈现高度抗性.经Western blot和RT-PCR检测发现,抗病转基因小麦体内外壳蛋白基因在病毒感染后的表达水平出现明显下降,认为所获得转基因小麦的抗性是由病毒诱导的基因沉默引起的.
The plant expression vector containing the coat protein gene of wheat mosaic virus (HMCV) was constructed by using the Act1 promoter and the bar selectable marker for herbicide, and introduced into the wheat line by particle bombardment.The transgenic lines T0 and T1 were amplified by PCR and PCR-RFLP After the test, the resistance of the progenies of the positive plants (T2 generation) to disease resistance was tested and the results showed that the foreign genes can be inherited in the offspring, and the progeny of one of the transgenic lines (P8-T2) The results of Western blot and RT-PCR showed that the expression level of coat protein gene in transgenic wheat after the virus infection decreased significantly, and the resistance of the obtained transgenic wheat was considered to be caused by virus-induced gene silencing caused.