Fuente: WIPO "tobacco agriculture" The invention discloses a transcriptional regulation gene NtMYB3-LIKE for tobacco nicotine biosynthesis and application of the transcriptional regulation gene NtMYB3-LIKE. The nucleotide sequence of the transcriptional regulation gene NtMYB3-LIKE for tobacco nicotine biosynthesis is as shown in SEQ ID: No.1, and the coded amino acid sequence is as shown in SEQ ID: No.2. The invention further discloses a preparation method of the transcriptional regulation gene NtMYB3-LIKE for tobacco nicotine biosynthesis. The invention further discloses a cloning method of the nicotine synthesis regulatory gene NtMYB3-LIKE. The cloning method comprises the following specific steps: A, determining an NtMYB3-LIKE gene sequence; b, extracting tobacco RNA, and carrying out reverse transcription to obtain first chain cDNA; c, designing and synthesizing a specific primer according to an NtMYB3-LIKE gene sequence, and carrying out PCR (Polymerase Chain Reaction) amplification by taking cDNA (Complementary Deoxyribose Nucleic Acid) as a template; d, recovering and purifying a PCR product; and E, constructing an overexpression vector containing the NtMYB3-LIKE gene. And performing overexpression on the overexpression vector in tobacco through agrobacterium tumefaciens-mediated transformation to prepare a transgenic plant. The nicotine contents in the middle and lower leaves of the obtained transgenic plant are respectively reduced by 36-40% and 36-40% compared with the control group. Therefore, the tobacco NtMYB3-LIKE gene has an application prospect in the aspect of reducing the nicotine content in tobacco leaves.