转Cry1Ac-w基因抗虫玉米的获得及分子鉴定

Acquirement and Molecular Identification of Transgenic Insect-resistant Corn Expressing Cry1Ac-w Gene

为将改造合成的抗虫基因(Cry1Ac-w)导入玉米基因组,以玉米杂交组合PA×PB的胚性愈伤组织为外植体,用双丙氨磷作为抗性愈伤组织的筛选剂,通过农杆菌介导法获得了82个再生植株。经标记基因(bar)和目的基因(Cry1Ac-w)的PCR检测,其中19株为阳性,即为推断的转Cry1Ac-w基因抗虫玉米。对其中2株(w1和w2)进行目的基因单酶切的Southern杂交分析,发现w1和w2分别为单位点和双位点插入,这也进一步证实目的基因已经整合入玉米基因组中。用Bt-Cry1Ab/Ac金标免疫试纸条进行蛋白质表达的检测,初步证实目标Cry1Ac-w蛋白在玉米中得到表达。

The purpose of this study was to introduce the modified Cry1Ac-w gene into the corn. Eighty-two regeneration plants were obtained through Agrobacterium-mediated method using embryo callus of the hybrid combination PA × PB as explants and the bialaphos as screening agent,but nineteen plants were putative transgenic plants,confirmed by PCR method of labelled gene（ bar） and target gene（ Cry1Ac-w）. Southern hybridization test（ single restriction enzymes digestion） showed successful integration of the foreign Cry1Ac-w gene into the corn genome and the w1 and w2contained one copy and two copies of target gene,respectively. The expressing of Cry1Ac-w protein was confirmed by Bt-Cry1 Ab / Ac immune detection strips.