Manipulation Of Lignin Biosynthesis Of Transgenic Poplar By 4CL Gene

Lignin is, second to cellulose, the most abundant organic compound in the terrestrial biosphere. In different tree species, lignin content varies between 15 and 36 % of the dry weight of wood. Lignin is a major constituent of cell walls of fibers and tracheary elements and provides these cells rigidity for structural support and impermeability for water transport. For the production of high-quality paper, lignin is considered as a negative factor because it must be extracted from the cellulose fraction by energy-requiring and polluting methods. For this reason, there is considerable interest in modifying lignin by genetic engineering to improve its extractability from wood. 4-coumarte: coenzyme A ligase (4CL) is a key enzyme, which decided the last step of lignin biosynthesis. Douglas's Group in UBC, Canada isolated whole cDNA fragment of 4CL genes, Ptd4CL3, from Populus trichocarpa x P. deltoides, whose proteins catalyze the CoA ligation of hydroxycinnamic acids, generating activated phenolic precursors for lignin biosynthesis, while Ptd4CL3 is strongly expressed in stem and is thought to be devoted to lignin biosynthesis in developing xylem tissue. The ultimate goal of our research is to obtain transgenic poplars with low-lignin contents for commercial paper industry and environmental purposes. Using this whole cDNA of Ptd4CL3, we undertook downstream work genetic engineering. We obtained these research results as followed as those: (1) Approximately 1.6kb fragment of Ptd4CL3 cDNA were inserted behind CaMV 35S promote of vector pBIN19/RT101 in an antisense or sense orientation to generate the binary vector pBIN19/RT101-AS4CL and pBIN19/RT101-S4CL. The binary vector was successfully mobilized into A. tumefaciens strain GV3101 (C58/pMP90). (2) A efficient leaf disc in vitro regeneration system has been optimized and established for five poplar cultivars with great economic interest in China, P. canadensis cl. 'Neva', P. canadensis cl 'Guariento', P. canadensis cl. 'Bellotto', P. deltoides x P. alba cl. 'Mincio'and P. deltoides x P. maximowiczii cl. 'Eridano', which are hybrid of section Aigeiros. Using this procedure of Agrobacterium-mediated transformation and leaf disc regeneration of transgenic plants, five poplar cultivars were transformed, and the transgenic plants were screened using Kan in root induced-media. (3) Transgenic plants of 6 poplar cultivars confirmed by PCR were transferred into soil and grown in the greenhouse for further characterization. Developing xylems were collected for 4CL enzyme assay from ten-month-old transgenic plants of P. deltoides x P. maximowiczii cl. 'Eridano'and P. tremula ×P. alba cl. 'INRA 717-1-B4'. 4CL enzyme activity in developing xylem was reduced from 10 to 50 % in the majority of the transgenic lines, which is in agreement with the result that the expression of 4CL was suppressed in the xylem of transgenic plants. The lignin content of these transgenic plants reduced by 10 %~40 % without abnormal morphogenesis and growth compared to that in the controls. RT-PCR analysis of transgenic plants of "INRA717"suggested that introduced sense and antisense 4CL suppressed transgenic and endogenous 4CL expression with stronger role of antisense than sense gene.