Abstract
This study investigated the spectral changes in alfalfa molecular structures induced by silencing of Transparent Testa 8 (TT8) and Homeobox 12 (HB12) genes with univariate and multivariate analyses. TT8-silenced (TT8i), HB12-silenced (HB12i) and wild type (WT) alfalfa were grown in a greenhouse under normal conditions and were harvested at early-to-mid vegetative stage. Samples were free-dried and grounded through 0.02 mm sieve for spectra collections with attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Afterwards, both univariate and multivariate analyses were conducted on amide, carbohydrate and lipid regions. Univariate results showed that silencing of TT8 and HB12 genes affected peak heights of most total carbohydrate (TC) and structural carbohydrate (STC), and structural carbohydrate area (STCA) in carbohydrate regions; and β-sheet height, amide areas, and ratios of amide I/II and α-helix/β-sheet in amide region; and symmetric CH2 (SyCH2), asymmetric CH2 (AsCH2) and (a)symmetric CH2 and CH3 area (ASCCA) in the lipid region. Multivariate analysis showed that both hierarchy cluster analysis (HCA) and principal component analysis (PCA) clearly separated WT from transgenic plants in all carbohydrate regions and (a)symmetric CH2 and CH3 (ASCC) lipid region. In the amide region, PCA separated WT, TT8i and HB12i into different groups, while HCA clustered WT into a separate group. In conclusion, silencing of TT8 and HB12 affected intrinsic molecular structures of both amide and carbohydrate profiles in alfalfa, and multivariate analyses successfully distinguished gene-silenced alfalfa from its parental WT control.
Highlights
Fourier transform infrared (FTIR) spectroscopy is an analytical technique that uses a polychromatic light source allowing for simultaneous collection of spectral absorption data from a wide range of electromagnetic spectra [1]
Three of the four major total carbohydrate (TC) peak heights were affected by alfalfa transformation with Testa 8 (TT8) and Homeobox 12 (HB12) RNAi constructs
There were no significant differences within transgenic alfalfa genotypes, TC1 peak height was decreased in transgenic alfalfa plants compared to wild type (WT) (p = 0.003)
Summary
Fourier transform infrared (FTIR) spectroscopy is an analytical technique that uses a polychromatic light source allowing for simultaneous collection of spectral absorption data from a wide range of electromagnetic spectra [1]. The reflection of IR beam creates an evanescent wave (4~6 μm) which can penetrate the sample on the crystal surface [7,9]. This technique has been used to detect the molecular changes induced by genetic modifications. Secondary protein structures were analyzed in this project, and α-helix/β-sheet height ratio was found higher in dual-transgenic alfalfa. Our group generated two genotypes of transgenic alfalfa, TT8i and HB12i, with silenced TT8 and HB12 genes, respectively. A pilot study previously reported on the carbohydrate structural features and the structural-nutrition relationships in TT8i and HB12i [15,16]. We used two multivariate analyses, hierarchical cluster analysis (HCA) and principle component analysis (PCA), on all spectral structural regions in an attempt to distinguish different genotypes
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