| Lignin is the second most abundant plant biopolymer mainly present in the secondary walls of tracheary elements and fibers, which allowing mechanical support, risistent to pathogen and efficient conduction of water and solutes over long distances within the vascular system. Lignin also has an important economic value, as it involved in the industrial and eviromental applications including paper making, foundry, building, energy and so on. Thus, the resolvation of molecular mechanism that controlling the biosysthensis of lignin is helpful to the applications of woody plants in both scientific and industrial areas.By recent studies, people demonstrated that the biosysthesis of monolignin is catalyzed by a set of enzymes and and requires a coordinated transcriptional activation of the genes involved in the lignin biosynthesis. Many transcription factors, including the MYB family members, have been shown to regulate lignin biosynthetic pathway and its deposition in woody tissues in various plant species through direct and indirect ways.Poplar is a widely planted specie of woody trees across the whole world, and valued by its significant applications in the industry of building and paper making. Since the the whole genome sequencing is achieved, Populus trichocarpa, as the genetic model system of woody trees, is used to reveal the molecular mechanism of lignin systhensis in vivo. Therefore, constructing the basic transcriptional network of lignin biosynthesis has significant economic and scientific research value. While the research of MYB transcription factors in populous is benefical for revealing the lignin-regulated network, and lay the foundation of industrial production and agricultural planting.In this study, we indentified PtrMYB092and PtrMYB152as novel TFs participate in the regulation of lignin biosysthensis pathway base on the bioinformatic analysis. To further understanding their bio-function and molecular mechanism, we designed and performed experiments as following:1. Identification and clone PtrMYB092/152genes from poplar genome and genetic transformation. To determine their functions, the coding regions and promoters of PtrMYB092and PtrMYB152are cloned from the genome of Populus trichocarpa and comfirmed by sequencing. Vectors for overexpression PtrMYB092and PtrMYB152in plant and analysisthe activity of thier promoters are constructed, as well as transformed into the Chinese white poplar (P. tomentosa Carr), Arabidopsis and yeast. The positive transformats are identified and confirmed by thier antibiotic resistant and PCR.2. PtrMYB092and PtrMYB152are specifically high expressed in xylem. The expression profiles of PtrMYBs cDNAs were investigated in various tissues of P. Trichocarpa with semi-quantitative reverse-transcription (RT) PCR and quantitative real-time PCR analysis demonstrated that PtrMYB092and PtrMYB152were expressed in various tissues and the highest level of expression was observed in xylem, root, petiole and stem. The histochemical studies of GUS activity revealed that, promoters of PtrMYB092and PtrMYB152are mainly active in the vascular elements of all organs. The GUS expression was also observed in primary xylem, deutoxylem, intrafascicular cambium, and fibers by microscopic observation of the stem sections.3. PtrMYB092and PtrMYB152are transcriptional factors activate the expression of their targets. In order to reveal the subcellular localization of PtrMYB092and PtrMYB152are in cells, PtrMYB092-GFP and PtrMYB152-GFP fusion proteins expressed in onion epidermal cells. The MYBs fussion GFP signals were found only in the nucleus, which indicate their function in transcriptional regulation. By the same time, the PtrMYB092and PtrMYB152were fused to the GAL4DNA-binding domain and introduced into yeast one hybrid system. The expression of the LacZ reporter genes suggest the MYBs are capable of activating the transcription of their targets.4. PtrMYB092and PtrMYB152up-regulated the expression of genes in lignin biosysthesis and regulation network. In order to investigate the effection of PtrMYB092and PtrMYB152overexpression in the expression of enzyme coding genes involved in the specific branch toward monolignol biosynthesis, quantitative real-time PCR analysis demonstrated that the expression of genes involved in the lignin biosynthesis were induced obviously. We also investigate the effection of PtrMYB092and PtrMYB152overexpression in the expression of other transcriptional factors in the network control secondary eall formation and xylem development, the results indicated that some of these genes were also induced obviously.5. Overexpression of PtrMYB092and PtrMYB152lead to ectopic deposition of lignin. With Phloroglucinol-HCl staining of the transgenic plants stem section and chemical analysis of lignin composition showing that, a significant higher accumulation of lignins was detected in transgenic plants harboring the PtrMYB092and PtrMYB152overexpression construct, compared to the wild-type control. Ectopic deposition of lignin was observed in phloem and vessel elements. Also, the lignin content of transgenic plants are significantly increased.All findings indicated that, PtrMYB092and PtrMYB152are transcriptional activators which promote the lignin biosynthesis through up-regulate the expression of enzymes and regulaters. Our researches may facilitate the further investigation and applications of woody plants and help the breeding of transgenic poplar strains. |
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