Department of Animal and Poultry Science, College of Agriculture and Bioresources, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N5A8, Canada.
London Research and Development Centre, Agriculture and Argi-Food Canada, 1391 Sandford Street, London, ON N5V 4T3, Canada.
Int J Mol Sci. 2018 Mar 31;19(4):1046. doi: 10.3390/ijms19041046.
This study investigated the spectral changes in alfalfa molecular structures induced by silencing of Transparent Testa 8 () and Homeobox 12 () genes with univariate and multivariate analyses. -silenced (TT8i), -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 and 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.
本研究通过单变量和多变量分析，研究了沉默 Transparent Testa 8（TT8）和 Homeobox 12（HB12）基因后紫花苜蓿分子结构的光谱变化。TT8 沉默（TT8i）、HB12 沉默（HB12i）和野生型（WT）紫花苜蓿在温室中正常条件下生长，在营养生长中期收获。样品经自由干燥后，通过 0.02mm 筛粉碎，用于衰减全反射傅里叶变换红外（ATR-FTIR）光谱收集。之后，对酰胺、碳水化合物和脂质区域进行了单变量和多变量分析。单变量结果表明，沉默 TT8 和 HB12 基因影响了碳水化合物区域中大多数总碳水化合物（TC）和结构碳水化合物（STC）以及结构碳水化合物面积（STCA）的峰高；酰胺区域中β-折叠高度、酰胺面积以及酰胺 I/II 和α-螺旋/β-折叠的比值；脂质区域中对称 CH2（SyCH2）、不对称 CH2（AsCH2）和（不对称）CH2 和 CH3 面积（ASCCA）。多变量分析表明，层次聚类分析（HCA）和主成分分析（PCA）都能清楚地将 WT 与转基因植物在所有碳水化合物区域和（不对称）CH2 和 CH3（ASCC）脂质区域区分开来。在酰胺区域，PCA 将 WT、TT8i 和 HB12i 分为不同的组，而 HCA 将 WT 聚类为一个单独的组。总之，沉默 TT8 和 HB12 影响了紫花苜蓿酰胺和碳水化合物图谱的内在分子结构，多变量分析成功地区分了基因沉默的紫花苜蓿与其亲本 WT 对照。
