草莓多聚半乳糖醛酸酶基因RNAi植物表达载体的构建及表达鉴定

Construction of Expression Vector of Polygalacturonase Gene from Strawberry for RNA Interfering and Identification of Expression

从草莓叶片中克隆到多聚半乳糖醛酸酶基因的1个281bp片断,构建含该正、反向互补重复序列DNA片段的中间载体,经测序证实连接正确后,克隆到植物表达载体p2300121中的GUS基因位置并经双酶切证实,得到RNAi表达载体。采用直接转化法将PG2300121导入根癌农杆菌菌株EHA105,并用新构建的工程菌对普通烟草进行了遗传转化研究。在Kanamycin选择压力下获得的烟草转化不定芽和完整植株,经PCR方法鉴定,证实了该基因已导入烟草基因组中。

A 281bp fragment of the Polygalacturonase gene was cloned from strawberry leaves. A vector that contained the sense and anti-sense orientatio inverted-repeat DNA fragment was constructed. After sequencing demonstration,the inverted-repeat DNA fragment was cloned into p2300121vectors. The RNAi expression vector was constructed. Moreover,PG2300121 was transferred into agrobacterium tumefacien EHA105 by direct DNA transfer. Genetic transformation was studied in Nicotiana tabacum,with the new engineering bacterium. The adventitious shoots and plants of Nicotiana tabacum was obtained under the selection pressure of Kanamycin PCR of genome of Nicotiana tabacum,showed that the target gene was transferred into the plants.