Genetic Manipulation Of Lignin Biosynthesis In Poplar

Lignins are aromatic polymers that are present mainly in secondarily thickened plant cell walls,which play important roles in plants but represent a major obstacle in chemical pulping,forage digestibility,and processing of plant biomass to biofuels. Thus there is of significantly economical and environmental value on dissecting complex biosysthetic pathways and engineering polygenic agronomic traits in plants. Several genes involved in the lignin biosynthetic pathways,including F5H,4CL,CAD and CCoAOMT,were studied by gene overexpresion and down-regulation in this paper.The main results are as follows:1.Transgenic Chinese white poplar(Populus) with 4CL-suppressing kept showing the present of transgene after growing in field for 4 years by Southern blotting analysis;The decrement of lignin content was maintained by Klason lignin content evaluation;Plants with 4CL gene down-regualtion have little shift in lignin compositional ratio(S/G ratio).Light micrographs of stem transverse sections revealed that vascular tissue of transgenic plants was in good condition,except that there were a few vessels of slight deformation or dysplasia,showing as smaller circumference,compressed shape,lined with normal vessels or being in clusters. These results imply that it is a good way to suppress the gene of 4CL to improve chemical pulping,and biofuels processing of poplar.2.The analysis of the Klason lignin content and thioacidolysis methods showed that lignin contents decreased in varying degrees in dual-gene transgenic plants with sense F5H and antisense CCoAOMT,and lignin components S/G ratio increased in varying degrees.This also indicates that dual or multi-gene regulation of lignin biosynthetic pathway might modify lignin properties more efficiently to create more feasible plants for industrial and agricultural production.3.Two full-length cDNA sequences named F5H and CAD were isolated by RT-PCR,as well as C4H promoter sequence were harvested by PCR with 1.1kb length in hybrid poplar(Populus tremula×Populus alba);the vetor which harbored respectively sense F5H or antisense CAD driven by C4H promoter was constructed, namely pC4H-F5H and pC4H-CAD.We used Agrobacterium to cotransfer dual genes into tobacco and obtained 48 independent kanamycin-resistant transgenic plants based on PCR analysis;with the same cotransfermation system and PCR analysis,22 independent kanamycin-resistant transgenic hybrid poplar plants were harvested.The use of culture medium MSA accelerated the growth of seedling,to reduce the period of tissue culture.Dual-gene transgenic tobacco and hybrid poplar plants are being identified and tested physiologically.