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Studies On The Regulation Of Lignin Biosynthesis By Antisense RNA

Posted on:2005-06-06Degree:MasterType:Thesis
Country:ChinaCandidate:J LuFull Text:PDF
GTID:2120360122493519Subject:Molecular and Cellular Biology
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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 two lignin related genes CCoAOMT and 4CL expression on lignin biosyntheisis were studied using antisense RNA techniques in this paper. The following presents the main progress:1 Two antisense expression vectors containing the cDNA of CCoAOMT or 4CL genes were transferred into Chinese white poplars mediated by Agrobacterium tumefacience. PCR-Southern analysis indicated that antisense cDNAs integrated into the genome of the transgenic poplars. The antisense genes expressed at transcriptional and translational level displayed by RT-PCR and Western blotting analyses. Klason lignin assay showed that both CCoAOMT and 4CL could control lignin content and led to the lignin reduction in transgenic poplars finally, without any obvious differences compared with the wild type on plant growth and carbohydrate synthesis. What most interested us was that the stems of transgenic poplars with marked lignin reduction turned brown-red. And 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 eight transgenic poplars with antisense CCoAOMT showed more than 10% reduction in lignin content, among which the most reduction was up to 26.20%. And three antisense 4CL lines reduced more than 10% lignin content, with the most reduction of 41.73%. These data laid a solid foundation of eliminating pollution resources produced by pulping industry.2. To effectively regulate lignin biosynthesis in plants, a C4H promoter fragment(the accession number: AY351673 ) was isolated from genomic DNA of Populustomentosa by PCR. Sequence analysis showed that this fragment had 1117 base pairs andshared 94% homo Logy with C4H gene's 5' untranslated region isolated from aspen {P.kitakamiensis). It contained CACCAACC and GTTAGGTT elements, which could berecognized by MYB-like transcriptional factor and Myb26 respectively. These conserved sequences were consistent with the biological functions in the phenylpropanoid pathway. 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 tobaccos, 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 tobaccos. Experiments using transgenic plants demonstrated that the expression of the fusion gene could effectively reduce lignin content in plants without any pronounced effects 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.
Keywords/Search Tags:lignin biosynthesis, antisense RNA, C4H, promoter, pulping
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