KBControl_KB693_KB6336_gTMT.txt

This study aimed to analyze the <i>γ-TMT</i> (<i>γ-tocopherol methyltransferase</i>) gene sequence and to perform <i>in silico</i> characterization of <i>γ-TMT</i> protein associated with tocopherol biosynthesis in mutant rodent tuber plants. Degenerate primers were designed based on the homologous <i>γ-TMT</i> gene sequences from several monocot species. Amplification of the <i>γ-TMT</i> gene produced a ±1300 bp DNA band in the mutant plants, while only a ±100 bp band was detected in the wild-type. The amplified region partially covers the <i>γ-TMT</i> gene, which has undergone mutations due to a combination of somaclonal variation and gamma irradiation. Thirteen base substitutions and four base deletions were identified between wild-type and mutant sequences. Comparisons between the mutants revealed five base substitutions and three base deletions. Translation analysis showed that KB 3-3-6 mutant produced a 406-amino acid protein, while KB 6-9-3 mutant produced a 407-amino acid protein. In contrast, the wild-type only translated into a 19-amino acid fragment. The identified exon region consisted of 354 and 355 amino acids in KB 3-3-6 and KB 6-9-3 mutants, respectively, while the amplified wild-type region was entirely exonic. Predicted protein structures based on the CDS sequences revealed notable differences in helix and loop orientation, particularly in the C-terminal domain and central regions of the protein. The protein structure of KB 6-9-3 mutant was more stable, with an RMSD value of 0.424 Å compared to 1.359 Å in KB 3-3-6 mutant. These structural differences are potentially linked to higher tocopherol content and enhanced anticancer activity in the mutant plants. This study provides foundational insights into the expression of the <i>γ-TMT</i> gene with tocopherol biosynthesis and supports the development of specific molecular markers in <i>Typhonium flagelliforme</i>.