Gene-Transformation-Induced Changes in Chemical Functional Group Features and Molecular Structure Conformation in Alfalfa Plants Co-Expressing Lc-bHLH and C1-MYB Transcriptive Flavanoid Regulatory Genes: Effects of Single-Gene and Two-Gene Insertion.

( L.) genotypes transformed with and transcription genes were developed with the intention of stimulating proanthocyanidin synthesis in the aerial parts of the plant. To our knowledge, there are no studies on the effect of single-gene and two-gene transformation on chemical functional groups and molecular structure changes in these plants. The objective of this study was to use advanced molecular spectroscopy with multivariate chemometrics to determine chemical functional group intensity and molecular structure changes in plants when co-expressing and transcriptive flavanoid regulatory genes in comparison with non-transgenic (NT) and AC Grazeland (ACGL) genotypes. The results showed that compared to NT genotype, the presence of double genes ( and ) increased ratios of both the area and peak height of protein structural Amide I/II and the height ratio of α-helix to β-sheet. In carbohydrate-related spectral analysis, the double gene-transformed genotypes exhibited lower peak heights at 1370, 1240, 1153, and 1020 cm compared to the NT genotype. Furthermore, the effect of double gene transformation on carbohydrate molecular structure was clearly revealed in the principal component analysis of the spectra. In conclusion, single or double transformation of and genes resulted in changing functional groups and molecular structure related to proteins and carbohydrates compared to the NT genotype. The current study provided molecular structural information on the transgenic plants and provided an insight into the impact of transgenes on protein and carbohydrate properties and their molecular structure's changes.