Characterization Of Transgenic Rice Plants Over-produced Fungi Cellulases And Suppressed Native Os4CL4 For Improving Lignocellulose Features And Enhancing Biomass Enzymatic Saccharification

Crop straws provide enormous biomass residues for biofuels and chemical production with less net carbon release.In particular,lignocellulosic ethanol has been regarded as a desirable biofuel.In principle,lignocellulose process involves in three major steps: initial physical and chemical pretreatment for cell wall destruction,sequential enzymatic hydrolysis for soluble sugars release and final yeast fermentation for ethanol production.However,due to lignocellulose recalcitrance,current biomass conversion is unacceptably costly.Hence,genetic modification of plant cell walls has been considered as a promising solution to biomass recalcitrance.In this study,we selected transgenic rice lines either over-expressing three types of fungi cellulases genes or silencing native 4-coumaric acid coenzyme A ligase(4CL)gene,and then examined cell wall compositions and biomass enzymatic saccharification under various chemical pretreatments in the transgenic rice plants.The major results were described below.1.Genetic modification of cellulose features by overexpressing heterologous fungi cellulases genes in transgenic rice lines: Using rice constitutive rbc S promoter and two signals for targeting to apoplast and chloroplast,this study respectively selected transgenic rice lines that over-expressed three Trichoderma reesei cellulases(EG II,CBH?,BGL I).(1)Compared to control(ZH11/wild type),the transgenic rice lines had relatively shorter plant height,but showed significantly improved lodging resistance;(2)The transgenic rice lines showed significantly reduced cellulose features(Cr I,DP),leading to much increased biomass porosity;(3)Major features of hemicelluloses and lignin were also altered to some degree in the transgenic rice lines;(4)Under mild chemical pretreatments,the transgenic lines had a complete biomass enzymatic saccharification,but the control plants only showed 70% saccharification;(5)The transgenic lines had the highest bioethanol yield at 21%(%dry matter);(6)A hypothetic model was proposed to highlight how major cellulose features were improved for largely enhanced biomass sacchsrification and bioethanol production in the transgenic rice plants.2.Genetic alteration of lignin monomer proportion by RNAi silencing native 4CL gene in transgenic rice plants: As 4-coumaric acid coenzyme A ligase(4CL)is a major enzyme involved in lignin biosynthesis,this study selected transgenic rice lines that silenced 4CL gene using RNAi method,and then determined 4CL enzyme activity in vitro and lignin constituent of transgenic rice plants.Compared to the control cultivar(NPB/wild type),the transgenic rice lines showed much reduced H-monomer proportion for largely enhanced biomass enzymatic saccharification under chemical pretreatments.Meanwhile,the Os4CL4 enzyme of the transgenic rice lines was examined to use more 4-coumaric acid than ferulic acid as substrate for lignin biosynthesis,consistent with increased H-monomer in the transgenic rice plants.