Fuente: WIPO "tobacco agriculture" The invention discloses genes capable of regulating and controlling nicotine content of a tobacco and application of the genes. A sequence of the genes capable of regulating and controlling the nicotine content of the tobacco is any one as shown in SEQ ID: No.1-21. The application of the genes is as follows: the genes capable of regulating and controlling the nicotine content of the tobacco are used for regulating synthesis and translocation amount of nicotine of the tobacco, so as to realize regulating and controlling of nicotine content in the tobacco. 21 genes obtained in the invention can form a stable hairpin structure, and analysis on the obtained tobacco non-coding small RNA sequence shows that part of small RNAs are from a sequence of a stem of the tobacco; and the small RNAs predict that target genes of the small RNAs are respectively targeted to a tobacco nicotine synthesis and transportation key protein coding gene. An miRNA regulating and controlling mechanism obtained in the invention can provide a deeper understanding for a role played by a key gene for a synthesis and translocation route of nicotine in tobacco, so that nicotine content character of the tobacco can be better improved.