【摘要】 根据植物转基因载体的特点和局限性,利用几种商业化载体进行克隆、酶切和重组,从而重构方便快捷的转基因植物表达载体。首先用PCR方法从pCAMBIA 1301质粒中扩增到含启动子CaM35S、完整GusA基因和终止子NOS三部分的片段,经TA克隆重组入pGM-T载体中获得pGM-35S-GUS-NOS重组质粒。然后对来源于新疆沙冬青的抗寒基因AnGPAT片段和pGM-35S-GUS-NOS重组质粒进行双酶切,连接获得中间载体pGM-35S-GPAT-NOS。再对中间载体和双元真核表达载体pCAMBIA 2300双酶切后连接,转化入大肠杆菌和农杆菌。经PCR、酶切及测序鉴定后确认得到改造后的植物表达载体p2300-35S-GPAT-NOS。结果表明,这是一种简便、高效、通用、经济的载体重构策略。更多还原

【Abstract】 According to the characteristics and limitations of plant transgene vectors,the and transgenic plant expression vectors were reconstructed by cloning,digestion and recombination in the base of the existing commercial vectors.First,design primers and amplify the gene fragments which were composed of CaM35S promoter,gusA gene and NOS terminator three parts from pCAMBIA 1301 plasmid.Recombinate the gusA gene fragments vector into pGM-T to get pGM-35S-GUS-NOS recombinant plasmid by TA cloning.Second,double digeste on cold resistance gene AnGPAT of and the pGM-35S-GUS-NOS plasmid,then connected to get intermediate vector pGM-35S-GPAT-NOS.Third,the intermediate vector and the dual eukaryotic expression vector pCAMBIA 2300 were double digested and connected to get the plasmid p2300-35S-GPAT-NOS.At last,transformed the plasmid p2300-35S-GPAT-NOS into E.coli and Agrobacterium.After PCR and restriction analysis,the transformation plant expression vector p2300-35S-GPAT-NOS was obtained.Experiments show that it is a simple,efficient,universal and economic reconstruction strategy for plant transgene vectors.更多还原