Fuente: WIPO "tobacco agriculture" The invention discloses a tobacco nicotine content regulation gene TIFY6B as well as a cloning method and application thereof. The nucleotide sequence of the tobacco nicotine content regulation gene TIFY6B is shown as SEQ ID NO:1; the amino acid sequence of the encoded protein is shown as SEQ ID NO:2. The cloning method comprises the following steps: synthesizing tobacco leaf cDNA, namely extracting tobacco leaf total RNA, and performing reverse transcription to obtain a first chain cDNA; performing PCR amplification on the gene TIF6B, namely taking the tobacco leaf cDNA as a template, designing a primer according to the sequence of the gene TIFY6B, performing PCR amplification, recovering and purifying the PCR amplification product, and sequencing. The invention further discloses application of the tobacco nicotine content regulation gene TIFY6B in an aspect of obtaining high-nicotine content transgenic tobacco plants. The expression of the gene TIFY6B is inhibited in the tobacco plant, the content of nicotine in tobacco leaves can be obviously improved, and the gene TIFY6B has wide application prospects in actual production.