Abstract: The N-demethylase gene CYP82E4 is a key gene responsible for the conversion of nicotine to nornicotine in tobacco. To develop breeding materials for cigars with a low nicotine conversion rate, EMS mutants of CYP82E4 gene were screened from an EMS mutant library of the cigar variety “Yunxue 39”. Through pooled amplification followed by sequencing analysis, two mutants including Mu1456 and Mu1568 were identified. The nucleotide mutation in Mu1456 was C139T, resulting in an amino acid change of L47F. Mu1568 had a nucleotide mutation of G134A, leading to an encoded amino acid mutation of G45D. SIFT predicted that the two mutation sites exerted a severe impact on protein function. Molecular docking analysis revealed that both mutant sites were located at critical positions of the substrate access channel. The mutated residues were substantially larger than their wild-type counterparts, causing steric hindrance in the substrate channel and thereby impairing nicotine binding to the active site. Compared to the wild-type control, the nicotine conversion rates of the M₄ generation mutants Mu1456 and Mu1568 were reduced by 0.39 and 0.66 percentage points, respectively, while the reductions in the M₅ generation were 1.07 and 1.57 percentage points, respectively. These results demonstrated that mutation of the CYP82E4 gene could significantly reduce the nicotine conversion rate in cigar tobacco. This study provided valuable breeding resources for developing reduced-harm cigar varieties.

Key words: cigar, CYP82E4 gene, nicotine conversion rate, EMS mutant