Construction Of CCR Gene RNA Interference Expression Vector And Transformation Of Poplar

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.CCR:Cinnamoyl-CoA reductase is a key enzyme,which decided the last step of lignin biosynthesis.RNAi,as an important gene silencing methods,has a significant application to the research of gene function and genetic melioration.In order to select and establish the regeneration and transformation system of poplar,to investigate effects of RNAi in poplar lignin improvement and to found a new molecular breeding method in poplar,we systematically explored the characteristics and parameters in inheritable transformation system by Agrobacterium,and constructed the expression vector of pBI121+2F then Agrobactirium mediated transformation.The integration of targeted gene was proved by PCR analysis,finally,the lignin content was tested.The main results are summarized as follow:1,The total Poplar DNA were extracted following a modification of CTAB method with some adjustments,Fourth extron(340bp) fragment from genome of CCR gene have been amplified by PCR amplification.The positive oriention and negative oriention of the fragment is ligated into pUCCRNAi,expression vector pBI121+2F have constructed by gene engeneering measure which containing kanamycin selecting gene,such as digestion,purification,ligation and transformation.Then transformed it into Agrobactirium tumefaciens LBA4404.2,In vitro micropropagation and a efficient leaf disc regeneration system have been optimized and established for nanlin95,and transgenic plants were screened using Kan in root induced-media.(1) Take MS as basic mediation with different concentration of 6-BA & NAA,the system of nanlin95 tissue culture has been optimized.The optimum culture medium for differentiation was(MS+6-BA 1.0 mg/L+NAA 0.2 mg/L+Sucrose 20 g/L+Agar 8 g/L),and[1/2MS+NAA 0.01 mg/L+IBA 0.2 mg/L+Sucrose 15 g/L+Agar 8 g/L]was optimum for root induced-media.(2) Through orthogonal design,an efficient transformation and screening system of nanlin95 mediated by Agrobateriurn tumefaciens with pBI121 was developed.The system was 7 days preculturation,20 min Agrobacterium tumefaciens(OD₆₀₀=0.6) infection with 200μmol/L acetosyringone,and 2 days' co-infection,the critical kanamycin sensitive concentrations for inducing shoots and roots of the poplar were 50 mg/L and 30 mg/L respectly.(3) With the optimized system of Agrobacterium-mediated transformation,the aimed fragment was transformed and generated transgenic plants.3,Transgenic plants of nanlin95 cultivar confirmed by PCR were transferred into soil and grown for further characterization.Developing xylems were collected from three-month-old transgenic plants of nanlin95.The lignin content of transformed plants reduced by 20%～30%without abnormal morphogenesis and growth compared to that in the controls.Transgenic plants with RNAi fragment of CCR for poplar cultivars will not only give novel insight into regulatory aspects of the pathway and on the structure and biological roles of lignin,but also provide a picture of genetic engineering for modified lignin composition and content.