Transgenic Research On The Relationship Between Glycosyltransferases And Lignin Biosynthesis Of Poplar(Populus Tomentosa Carr.)

Glycosylation occurs in a wide range of biological processes in plants and is thought to play an important role in the production of a range of plant compounds. Glycosyltransferases (GTs) are enzymes responsible for glycosylation of plant compounds. The transfer of a sugar onto a lipophilic acceptor changes acceptor in its stability, chemical properties, bioactivity and enables the acceptor access to membrane transporter systems. Therefore, glycosyltransferases might have an important role in maintaining cell homeostasis and regulating plant growth and development.Lignin is aromatic polymers that are present mainly in secondarily thickened plant cell walls. It is synthesized from the oxidative coupling of p-hydroxycinnamyl alcohol monomers and related compounds. Lignin is covalently bound to celluloses and hemicelluloses and provides strength and rigidity to the cell wall, allowing plants to grow upward. It also plays important roles in improving the cellular transport capacity and resisting the infringement of pathogen microorganisms. But in the industrial and agricultural production, lignin imposes negative effects on the the fully use of cellulose because they are interwoven. Therefore, it has become a focus in recent years to use genetic engineering to change lignin content and biosynthesis.The previous studies suggested that glycosylation may play an important role in regulating the process of lignin biosynthesis. It is likely that the glycosylation could increase the solubility, stability, storage and transporter binding properties of lignin monomers, and it could also reduce the toxicity of lignin monomers on cells. Understanding the relationship between glycosyltransferases and lignin biosynthesis is therefore not only of scientific but also practical interest.Poplar is a woody plant of great commercial and ecological value. Its high lignin contents, clear genetic background and rapid growth make it an ideal model for studying the lignin biosynthesis. This work studied the relationship between three poplar glycosyltransferases and lignin biosynthesis through transgenic technology, and it was found that one glycosyltransferases, PtGT1, can significantly increase lignin contents when it was ectopically expressed in tobacco. This work would lay the foundation for further studies on the roles of glycosylation in regulating poplar lignin biosynthesis and for the lignin genetic engineering in poplar and other woody plants. The main contents and results of this study are listed as follows.1. Sequences of poplar glycosyltransferases PtGT1, PtGT2and PtGT3were analyzed.The deduced amino acid sequences of poplar PtGT1, PtGT2and PtGT3were aligned with Arabidopsis UGT72E1-E3. A phylogenetic comparison was made between PtGT1, PtGT2, PtGT3and all40published Arabidopsis family1GTs. The poplar PtGT1, PtGT2and PtGT3were found to be located on a unique branch with Arabidopsis UGT72E1-E3and it was also found that PtGT1, PtGT2and PtGT3exhibits relatively high identity to the Arabidopsis UGT72E1-E3. This suggests that the poplar PtGT1, PtGT2and PtGT3might be involved in the glycosylation modification of lignin precursors.2. The plant expression vectors of PtGT1, PtGT2and PtGT3were constructed. Transgenic tobacco and poplar were obtained.In order to study the effects of constitutive over-expression of PtGT1, PtGT2and PtGT3on plant growth and development, we first constructed the plant expression vectors of those genes. Then genetic transformation of tobacco and poplar plants was conducted using the Agrobacterium-mediated method. A series of analyses including antibiotic selection, PCR and RT-PCR demonstrated that the transgenic tobacco and transgenic poplar plants were obtained, thus providing the materials for the subsequent phenotypic and physiological analysis.3. Transgenic tobacco plants were analyzed and it was found that the over-expression of PtGT1resulted in significantly higher lignin contents and earlier flowering. The phenotypes and physiology of transgenic tobacco plants were investigated through lignin staining, Klason lignin content measurement, cellulose content analysis and the observation of visible phenotypes. It was found that the over-expression of PtGT1gene in tobacco resulted in significantly higher lignin contents and earlier flowering than wild type. However, the transgenic tobacco plants of PtGT2or PtGT3gene had no significant phenotypic or physiological changes compared to wild type plants.4. The expression pattern of the poplar PtGT1, PtGT2and PtGT3genes were studied, and the optimal explant for poplar tissue culture was identified.The expression levels of PtGTl, PtGT2and PtGT3genes were evaluated using RT-PCR technology for the poplar tissues of stems, leaves and roots. Experimental results showed that PtGTl transcripts were expressed at higher levels in upper stems but at lower levels in other tissues. However, the levels of PtGT2and PtGT3transcripts in the examined tissues had no significant difference.Three different explants of poplar:leaves, stems and petioles, were used for tissue culture and their differentiation and regeneration rates were compared in the same culture conditions. It was found that the petioles had the highest differentiation and regeneration rate, followed by stems and leaves. Thus, this experiment identified the optimal explant for the further poplar tissue culture and transgenic studies.In this study, although it was found that the poplar glycosyltransferase PtGTl had profound impacts on lignin biosynthesis and flowering time of transgenic tobacco, its roles played in poplar itself need to be further investigated. In addition, the physiological roles of PtGT2and PtGT3also remain to be answered in the next study.