甘蔗锌指蛋白ShZP基因正义反义植物表达载体的构建及转化烟草

摘要

According to the cDNA sequence of sugarcane zinc finger protein ShZP gene cloned in our laboratory, specific primers were designed in which the restriction sites were within them and linked outside the open reading frame. Through PCR amplification methods, the cDNA segments of ShZP gene's coding region 725 bp were forwardly and reversely inserted to the places between the Prd29A promoter and NOS terminator of the intermediate vector pCRBI. The sense plant expression vector pCRBIShZP and antisense plant expression vector pCRBIantiShZP were constructed. pCRBIShZP and pCRBIantiShZP were respectively introduced into Agrobacterium tumefaciems strains EHA105 through the freeze-thaw method and transformed into tobacco through agrobacterium-mediated transformation method. Twenty three resistant plants were obtained through 12 mg/L PPT successive screening. Among these plants, 3 transgenic sense gene tobacco plants and 4 transgenic antisense gene tobacco plants were confirmed by the specific fragment PCR. The specific fragments were partial region of promoter and partial region of target gene. The results of PCR proved that the ShZP gene, which was exogenous zinc finger proteins gene, had been integrated into the tobacco genome. This study made a good foundation for plant stress resistant genetic engineering.%根据本实验室克隆的甘蔗锌指蛋白ShZP基因cDNA序列,在开放阅读框两侧设计特异引物,通过PCR扩增引入相应的酶切位点,把甘蔗ShZP基因的编码区(大小为725bp)cDNA片段,分别以正向和反向插入到中间载体pCRBI的Prd29A启动子和NOS终止子之间,构建了其正义植物表达载体pCRBIShZP和反义表达载体pCRBIantiShZP.采用冻融法分别将pCRBIShZP和pCRBIantiShZP导入根瘤农杆菌菌株EHA105中,通过农杆菌介导法对烟草进行转化,经过12mg/L的PPT连续抗性筛选,共获得23株抗性植株,对其中的5株转正义基因烟草植株和9株转反义基因烟草植株进行PCR检测,分别获得3株转正义基因的阳性植株和4株转反义基因的阳性植株.PCR检测结果初步证明外源甘蔗锌指蛋白ShZP基因已成功整合到烟草基因组中,这一研究为植物抗逆基因工程研究打下了一定的基础.