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【目的】利用载体重组技术分别将高山离子芥冷诱导基因(Cbcor15a)和报告基因eGFP插入载体pCambia1300-bar,并引入玉米泛素基因启动子UBi-1代替载体本身启动子CaMV35s,重组为适合甘蔗转基因的二元植物表达载体pCambia1300-cbcor15a-bar。【方法】参照pCambia1300-bar载体多克隆位点和基因Cbcor15a、eGFP和启动子UBi-1的核苷酸序列设计引物,通过载体重组技术将基因和启动子分别插入相应的位点。利用基因枪分别将pCambia1300-bar载体和重组载体导入洋葱表皮细胞,用荧光显微镜和激光共聚焦显微镜观察。【结果】与导入pCambia1300-bar载体的洋葱表皮细胞相比较,导入重组载体pCambia1300-cbcor15a-bar的洋葱表皮细胞内有强烈的绿色荧光信号。【结论】重组甘蔗转基因二元植物表达载体启动子Ubi-1能够调控下游冷诱导基因Cbcor15a和报告基因eGFP的正常高效表达,为外源基因Cbcor15a转化甘蔗提供保障。
【OBJECTIVE】 The gene of Cbcor15a and reporter gene eGFP were inserted into vector pCambia1300-bar respectively by vector recombination technique, and the promoter UBi-1 of maize ubiquitin gene was introduced to replace CaMV35s promoter, which was recombined into sugarcane Transgenic binary plant expression vector pCambia1300-cbcor15a-bar. 【Method】 The primers were designed based on the multiple cloning sites of pCambia1300-bar vector and the nucleotide sequences of the genes Cbcor15a, eGFP and UBi-1. The genes and promoters were inserted into the corresponding sites respectively by vector recombination technology. The pCambia1300-bar vector and the recombinant vector were introduced into onion epidermal cells using a gene gun, respectively, and observed under a fluorescence microscope and confocal laser scanning microscope. 【Result】 Compared with onion epidermal cells introduced into pCambia1300-bar vector, the green epidermal cells of onion epidermal cells introduced into the recombinant vector pCambia1300-cbcor15a-bar showed a strong green fluorescence signal. 【Conclusion】 Ubi-1, a promoter of recombinant binary vector of transgenic sugarcane, can regulate the expression of downstream cold-induced gene Cbcor15a and reporter gene eGFP efficiently and provide a guarantee for the transformation of exogenous gene Cbcor15a into sugarcane.