| Traditional fossil fuels are non-renewable and environmentally polluted, and thus could potentially destruct the ecological environment, so the use of it should be strictly limited. As a result, future demand for renewable energy will increase significantly. Therefore, the use of alternative bioethanol or biofuels will gradually replace fossil fuels (oil, natural gas, coal). By doing so, it will heavily reduce dependence of related industries to fossil fuels, and it will help to alleviate all the correlated environmental problems. Poplar species is a preferred source for forest biomass research due to its wide distribution, strong adaptability, quick production, asexual reproduction and clear genetic background.Lignocelluloses are the largest renewable resource. However, the "biomass recalcitrance" created by tight binding of lignin, hemicelluloses and cellulose, which is also the major obstacle for cellulose-based ethanol. Moreover, the lignin within cell wall is a major contributor to pollution of paper production and high cost of cellulosic ethanol. Lignin content can be reduced and lignin components can be altered by inhibiting the expression of critical genes in lignin biosynthetic pathway, thus improving the conversion efficiency of cellulose-based ethanol and lowering its conversion costs.In this study, we cloned two genes encoding enzymes involved in lignin biosynthesis, coumaroyl shikimate 3-hydroxylase (C3H) and hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase (HCT), and constructed them into RNAi vectors. Poplar was transformed via the leaf-disc method. A total of 26 C3H1-RNAi transgenic lines and 9 HCT1-RNAi transgenic lines were obtained and vegetatively propagated by cutting for each lines in the greenhouse. The kanamycin-resistant plants were subjected to further analyses of target gene expression, cell wall components, and cell wall structure. Quantitatively real-time PCR (qRT-PCR) showed the decrease of transcript levels of C3H1 and HCT1 in RNAi transgenic lines, with a up to 75%reduction in analyzed C3H1-RNAi lines 10,55 and 57 compared with, and a up to 69%reduction in analyzed HCT1-RNAi lines 202,205 and 207. In the same growing period, the transgenic plants were shorter than the wild type obviously; when compared with the wild type, the roots of the transgenic plants were more flourishing, adventitious roots grew and the number lateral roots were larger obviously.Observation and comparison of the anatomical structure of transection of C3H1-RNAi, HCT1-RNAi transgenic plants and the wild type (84K), showed that in transgenic plants the cell wall thickness in secondary xylem decreased, while the number of tracheal vessel (TE) increased and the distribution of TE was irregular. The number of phloem fibers in transgenic plants was less than that in the wild type. Besides, the HCT1-RNAi transgenic plants have more xylem rays than the wild type, which indicated that the development of xylem and the pattern of lignin deposition were changed in transgenic plant. In addition, compared with the wild plants, some structures of the transgenic plants of C3H1-RNAi and HCT1-RNAi, including the average diameter of vessel cavity, the cell wall of vessels, the diameter of fiber cavity and the cell wall of fibers, become smaller. The ratio of cell wall to cavity was less than 1, indicating that both transgenic plants are fit to be as raw materials of papermaking.In the improvement of wood property, the quantitative 13C NMR,2D HSQC,31P NMR and gel chromatography (GPC) techniques were used to investigate the structural features of the lignin:(1) An improved method combined milled wood lignin (MWL) and enzymatic hydrolysis lignin (CEL) is used to extract lignin fractions from different locations of the plant cell walls. The results showed that down-regulated poplar lignin had higher β-O-4’ linkages and S/G ratios content compared to the control. The PB content in the transgenic lignin was increased. In addition, H-type lignin units could be found in the transgenic lignin; (2) EMAL samples were extracted from transgenic poplars with high yield and purity. The results demonstrated that the down-regulated poplar lignin had a higher β-O-4 linkages content, while the content of β-β and β-5 linkages in down-regulated poplar lignin was decreased. The higher content of β-O-4 linkages could facilitate the subsequent delignification process. Interestingly, the total OCH3 content in the HCT-EMAL and C3H-EMAL was decreased, while the corresponding S/G ratios was increased. (3) To better understand the structural changes of hemicelluloses caused by gene perturbation, the DMSO and alkaline hemicelluloses were extracted from transgenic and wild-type 84K, the 13C NMR,2D HSQC, and gel chromatography (GPC) techniques were employed. The results showed that the molecular weight of hemicelluloses extracted from C3H down-regulated poplar was higher than that of the control. In addition, the polydispersity (Mw/Mn) of the hemicelluloses from C3H down-regulated was narrower than that of hemicelluloses from CK poplars. Moreover, the hemicelluloses extracted with aqueous alkaline were mainly 4-O-methyl-D-glucurono-D-xylan, while DMSO-extracted hemicelluloses were composed of 4-O-methyl-D-glucurono-D-xylan and other heteroglycan. In short, C3H and HCT down-regulation can change the composition and structural features of the lignin, improve the chemical processing properties of wood and improve the extraction of hemicelluloses. The results lay the research foundation for genetic breeding research on improvement of wood material, high efficient utilization of energy type poplar. |
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