Transformation And Construction Of C4H Gene RNAi Vector And UGPase Gene Over-expression Vector On Populus

Lignin, a major component of secondary cell wall of vascular plants, has long beenrecognized for its negative impact on forage quality, paper manufacturing and cellulosicbiofuel production. Lignin quantity and reactivity are two major barriers to wood-pulpproduction. Over the last two decades study on phenylalanine metabolic p athway, it hasbeen clear to illustrate the main function of key enzyme participating in lignin biosynthesismetabolic. Cinnamate4-hydroxylase (C4H) is a cytochrome P450-dependentmonooxygenase that catalyzes the4-hydroxylation of coumaric acid. RNA interference(RNAi) is an effective tool for decreasing the expression of target gene on transcriptionallevels. In this study, we use methods of vector construction and transgene to generatetransgenic poplar suppressed in C4H expression, producing trees with low content oflignin.Cellulose microfibrils play essential roles in the organization of plant cell walls, whichare also needed components in wood pulping and bleaching process. It is remarkable howlittle is known about how it is synthesized. Scientists prefer to view cellulose synthesis as a“cellular process” rather than “enzyme mediated reactions”. At present, the study ofcellulose synthesis mainly focuses on cellulose synthase (CesA) and UDP-glucosepyrophosphorylase (UGPase). It had been proved that over-expression UGPase resulted ina significant increase in biomass and cellulose content. In this study, we usedover-expression and transgene methods to generated transgenic poplar enhanced inUGPase expression, producing trees with high content of cellulose.Populus×euramericana cv. Nanlin95and Populus deltoids cv. Nankang were used asplant material in our study. We constructed expression vector C4H RNAi andover-expression vector of UGPase, then Agrobacterium mediated transformation wascarried out subsequently. Finally, we obtained transgenic plants by PCR analysis. The mainresults were summarized as below:(1) A partial C4H gene was cloned from Populus×euramericana cv. Nanlin95byRT-PCR and C4H RNAi vector was successfully constructed. A completed UGPase genewas cloned from Populus deltoids cv. Nankang by RT-PCR with1410bp cDNA length,encoding469amino acids. Then, the construction of UGPase over-expression was carriedout subsequently.(2) By the analysis of bioinformatics, the predicted molecular weight was51.64KDand theoretical pI was5.56. EST expression analysis showed that UGPase maily expressed in flower or other terminal vegetative buds. The results of Pfam analysis showed that therewas a significant UDPGP domain in UGPase.3D-JIGSAW protein comparative modelingwas used to predict UGPase3D structure. The results of phylogenetic tree showed that theUGPase gene family in dicotyledon was less conservation than monocotyledon.(3) We successfully constructed UGPase gene over-expression vector pBI121+UGPase. By the methods of Agrobacterium mediated transformation, we finally obtained7resistant seedlings. Analyze by PCR indicated that there were5seedlings successfullytransformed UGPase gene.In general, my research successfully cloned partial encoding gene for C4H andcomplete encoding gene for UGPase, then successfully constructed C4H RNAi vector. Bythe analysis of various bioinformatics software, including sequence alignments, structureprediction and phylogenetic relationship, we had obtained comprehensive understanding ofUGPase gene family. Finally,5transgene plants were identified, providing a foundation forthe more research on lignin and cellulose synthesis metabilization.