| Lignin is a phenolic polymer and plays important roles in plants, but is the main resource of papermaking pollution. Thus there is of economical and environmental interests in cultivating materials with genetically modified lignin for pulping industry. The effects of downregulation of three lignin related genes COMT, CCoAOMT and 4CL expression on lignin biosyntheisis were studied in this paper. The following presents the main progress.1. To identify the function of two methyltransferases (COMT and CCoAOMT), two single antisense expression vectors containing the cDNA of COMT or CCoAOMT gene and one dual antisense expression vector containing cDNAs of both OMTs genes were transferred into tobacco mediated by Agrobacterium tumefacience. PCR-Southern analysis indicated that antisense cDNAs integrated into the genome of the transgenic tobacco. The antisense genes expressed at transcriptional level displayed by Northern dot analysis. Klason lignin assay showed that CCoAOMT could control lignin content more effectively than COMT. The latter was only specifically responsible for syringyl (S) lignin pathway. The antisense CCoAOMT was also transformed into Chinese white aspen (populus tomentosa). It inhibited the expression of the endogenous target gene at transcriptional and translational levels, and led to the lignin reduction in transgenic poplars finally. Till now eight transgenic lines with more than 10% lignin reduction were obtained, among which the most reduction was 26.20%. These data laid a solid foundation of eliminating pollution resources produced by pulping industry.2. The cDNA encoding 4-coumarate : CoA ligase (4CL) from Populus tomentosa was analyzed. RT-PCR and Northern dot analysis indicated that 4CL mRNA was highly accumulated in xylem and its expression revealed a biphasic pattern in one growing season, almost in phase with the formation of early wood and lately wood. This demonstrated that 4CL gene was involved in lignin biosynthesis. Transgenic researches displayed that the expression of antisense 4CL resulted in the decreasing of lignin content in transgenic tobaccos and Chinese white poplars with little effects on plant growth andcarbohydrate synthesis. But the stems of transgenic poplars with marked lignin reduction turned brown-red. The color darkness was positively related with lignin reduction degree, which could be used as an index for selecting poplars with lignin reduction. Till now three transgenic poplars with more than 10% lignin reduction were obtained, among which the most reduction could be up to 41. 73%.3. To effectively regulate lignin biosynthesis in plants, a C4H promoter fragment (the accession number: AY351673) was isolated from genomic DNA of Populustomentosa by PCR. Fluorometric and histochemical GUS analysis showed that the expression of a C4H-GUS fusion product was temporally and spatially specific. It was mainly expressed in the lignified tissues and its activity gradually increased from the first to nine internodes in stems of tobacco, preceding the lignin deposition. C4H-GUS expression was also inductive by wounding. C4H promoter fused to antisense CCoAOMT cDNA was used for manipulating lignin biosynthesis in transgenic tobacco. Experiments using transgenic plants demonstrated that the expression of the fusion gene could effectively reduce lignin content in plants without any pronounced effect on plant growth and the carbon allocation to carbohydrate pool. These results suggested that the C4H promoter from Populus tomentosa could be employed in genetically improving wood quality.4. To investigate the methylated pathway occurring in monocots, three CCoAOMT genes were firstly isolated from Zhonghua 10 (Oryza sativa L. ssp. japonica) and their gene structure and expression pattern were studied. Results suggested that three rice CCoAOMT genes were closely related with lignin biosynthesis. This opened up the possibility of improving the resistance of rice against lodging and forage digestibility by genetically modifying lignin biosynthesis.
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