Obtaining The Transgenic Poplars With Low Lignin Content And Cloning Of 4CL Promoter From Chinese White Poplar (Populus Tomentosa)

Lignin is one of the major secondary products in plants with important biological functions. However, in chemical process of paper pulping, lignin must be extracted from the cellulose fraction that is the material of papermaking with polluting and cost-increasing methods. The Chinese White Poplar (Populus tomentosd) is Chinese local specific tree and one of important resource tree species for papermaking. The purpose of this study is to regulate lignin biosynthesis at molecular level, reduce lignin content by antisense RNA in Chinese white poplar and finally produce resource tree species that is more suited for papermaking.Antisense CCoAOMT cDNA and 4CL cDNA had been transformed into triploid Chinese white poplar mediated by Agrobacterium tumefaciens. The transgenic plants were identified by PCR, PCR-Southern and Southern analysis. The antisense gene had also expressed indicated by RT-PCR and Western analysis. Klason lignin measurement of transgenic poplars growing for 6 months showed that repression of CCoAOMT and 4CL expression could result in remarkable reduction of lignin content in transgenic poplars. Repressing the expression of 4CL cDNA can result in a highest lignin reduction of 41.73% comparing with wild type. The highest reduction of lignin content in one of transgenic poplars expressing antisense CCoAOMT cDNA is 16.31%.Analysis of holocellulose content in antisense 4CL expressed transgenic plants showed that there is no significant difference between transgenic and wild poplars. We considered that 4CL may not be the metabolism control point between lignin and carbohydrate biosynthesis, which was different from the conclusion of the foreign studies.The stems of transgenic poplars displayed red-brown color with different shades after the bark was peeled, while those of untransformed plants were white. The reductions in lignin content of transgenic plants, which stems are complete red-brown, are comparable high. The lignin content of transgenic plants which stems are mottled red-brown is moderate reduced. Wiesner reaction of untransformed plant is typical purple-red, while that of complete red-brown stems exhibited red. Wiesner reactions of white parts in mottled red stems of transgenic plants displayed typical purple red like untransformed plants, while that of red-brown parts showed red color, which more testified the relationship of lignin content with stem color in transgenic plant. We consider that the stem color of transgenicplant could be employed as supplementary trait to screen the transgenic plant with reducedlignin content.4CL promoter fragment was amplified by PCR method from Populus tomentosa genome DNA.The sequence analyses show it shares 95% homology with 4CL promoter in P. tremuloid at nucleotide level.Through a series of DNA recombination, the expression vector containing 4CL promoter was constructed, it was confirmed by PCR and restriction analysis.4CL promoter drived expression cassette was transformed into tobacco mediated by Agribacterium tumefacience. We obtained transformed plants.