Preliminary Study On The Regulatory Network Of Lignin Biosynthesis Of Sorghum Bicolor(L.)Moench By SbbHLH1

With the depletion of fossil energy sources in near future,the possibility of the global energy crisis is increasing.Therefore,new alternative energy source must be developed to adapt rapid economic growth.Bioenergy is a renewable,highly efficient and clean green energy source.Plant celluloses were used as raw material to produce ethanol in two successive processes,saccharification and fermentation;ethanol can be directly added to gasoline as fuel ethanol.Sweet sorghum has become a new type of energy crop worldwide due to its high stress tolerance,high biomass and high sugar content in stems.However,due to much complex of plant cell walls and high content of lignin in stem,the efficiency of cellulose conversion to ethanol is much lower;all this has become an important restrict factor and big obstacle to bioethanol industry.One of the possible ways to overcome this problem is to reduce lignin content and increase cellulose content in raw material by change the expression of some key genes that regulate lignin and cellulose biosynthesis.In the future,genetic engineering must play an important role in improve the production of bioethanol.SbbHLHl is a negative regulator of lignin synthesis in sorghum(J Exp Bot.2013),but the mechanism of it is poor understood.In this research,the target genes of SbbHLHl were identified through EMSA by binding E-box in promoter,and interaction proteins were recognized by AF-MS,Y2H and BiFC.Two Arabidopsis OE lines(SbbHLH1-GFP)with low lignin content were further checked by Wiesner staining of stem and Q-PCR of some key genes in phenylpropane pathways,the results were further proved that SbbHLH1 is a negatively regulator to lignin synthesis.Sorghum leaf protein and Arabidopsis total protein were used to combine SbbHLH1-His recombinant protein by affinity chromatography-mass spectrometry(AS-MS),llpossible "interactive" proteins were further verified by Y2H and BiFC.Finally,one of the interaction protein cytochrome P450(AtCYP98A3)were confirmed to interaction with SbbHLH1.Due to AtCYP98A3 is the C3H gene in the lignin metabolism pathway,this interaction must affect the activity of these two proteins in somehow,and further influence the synthesis of lignin.Competitive binding experiments were further confirmed the binding site of SbbHLH1 to AtPALl promoter.We also found SbbHLH1 binding to the E-box of the 4CL1 promoter.Therefore,SbbHLHl control lignin synthesis at least through two levels that is transcription and proteins interaction.Together with SbLIM1,SbbHLH1 may be regulate or influence other transcription factors,that direct or indirect involved in lignin synthesis.Cellulose degradation experiments were conducted in the five-year seedlings of SbbHLHl transgenic Populus tomentosa to prove that low lignin was facilitates the degradation of cellulose by cellulase in poplars.The results show that lignin content was significantly negative correlated with the conversion rate of cellulose.All these maybe proved that new varieties of energy plants(include poplar)with low lignin content can be created by genetic engineering.