Fecha de publicación: 14/01/2020 Fuente: WIPO "tobacco agriculture" The invention discloses a tobacco nicotine synthesis regulatory gene NtERF91 as well as a cloning method and an application thereof. The nucleotide sequence of the nicotine synthesis regulatory gene NtERF91 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 the cloning method of the nicotine synthesis regulatory gene NtERF91.The cloning method comprises the following specific steps: A, determining an NtERF91 gene sequence; B, extracting tobacco RNA, and performing reverse transcription to obtain a first chain cDNA; C, designing and synthesizing a specific primer according to an NtERF91 gene sequence, and performing PCR amplification by taking cDNA as a template; D, recovering and purifying a PCR product; and E, constructing an overexpression vector containing the NtERF91 gene. An overexpression vector is overexpressed in tobacco through agrobacterium-mediated transformation, and a transgenic plant is prepared. The nicotine content of the obtained transgenic plant is improved by 60% or above compared with that of a control group. Therefore, the tobacco NtERF91 gene has a great application prospect in the aspect of cultivating tobacco with high nicotine content.