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以小麦品种西农1376为受体,利用Overlapping PCR的方式构建了防御素基因alfAFP和抗菌肽基因spCEMA融合基因的表达载体,与pAHC20共转化幼胚愈伤组织,获得转基因再生T0代植株269株,春化后移栽成活188株。对叶片基因组DNA进行目的基因和bar基因PCR扩增表明其中7株是共整合植株,共整合频率为0.1%,初步证明融合基因已成功通过共转化方式导入受体小麦品种中。影响共整合率的因素分析表明,在使用除草剂PPT对bar基因进行筛选的过程中,筛选时间的缩短并不会明显降低筛选的效率。
Using wheat cultivar Xinong 1376 as the receptor, the expression vector of defensin gene alfAFP and antimicrobial peptide gene spCEMA fusion gene was constructed by Overlapping PCR, and the embryo callus was co-transformed with pAHC20 to obtain 269 transgenic T0 plants , After transplanting live vernalization 188. The target genomic DNA and bar gene PCR amplification showed that seven of them were co-integrated plants with a total integration frequency of 0.1%. It was initially proved that the fusion gene was successfully introduced into recipient wheat varieties by co-transformation. The analysis of the factors influencing the rate of co-integration showed that the shortening of screening time did not significantly reduce the screening efficiency when the herbicide PPT was used to screen the bar gene.