Abstract: For revealing the function of CYP82E4v1 gene involving in nornicotine biosynthesis in Nicotiana tabacum L., an Agrobacterium tumefaciens-mediated transformation method was used to transform tobacco cv. K326 with two plant expression vectors, pLF-35S-CYP, which contained CYP82E4v1-overexpressing fragment, and pLF-35S-CYPi containing interfered expression fragment, respectively. Sixty-one transgenic plants were acquired, including forty-two overexpressing plants and nineteen interfered ones. The results of HPLC showed that the nornicotine content in CYP82E4v1-overexpressing plants was 88.9% higher than, that in interfered expression plants was 61.1% lower than that in the plants of cv. K326 (CK). It indicated that the overexpression of CYP82E4v1 increased nornicotine content in tobacco, whereas the interfered expression of CYP82E4v1 gene inhibited nornicotine biosynthesis. The results of Real-Time PCR analysis indicated that the expression level of CYP82E4v1 gene in overexpressing plants was over 20 times the level in the CK, while that in interfered expression plants was only 6% of that in the CK. Meanwhile, the other nornicotine biosynthesis genes, such as CYP82E5v2 and CYP82E10, were suppressed to different extents in interfered expression plants. In addition, the "Gene-deletor" system contained recombinase gene FLP driven by the promoter of Arabidopsis thaliana senescent gene SAG12, which deleted the exogenous gene GUS∷NPT II during the late stage of leaf development, was introduced into the mentioned expression fragments for the reason of acquisition of selectable-free transgenic tobacco plants with low nornicotine content.