Effects Of Driving VND7 On Secondary Walls In Arabidopsis Senescence And Effect Of CslF6 On Enzymatic Hydrolysis Efficiency Of Rice Biomass

The Arabidopsis VASCULAR-RELATED NAC-DOMAIN 7(VND7)is a key regulatory gene for the differentiation of protoxylem vessel.Studies have shown that expression of VND7 can result in thickening of the secondary walls of plants and ultimately increase biomass.A spatio-temporal strategy for the use of senescence-related gene promoters was proposed,which solved the problem that the transgenic plants were affected by the disorder of nutrient distribution and caused normal growth and development.In this study,based on the idea of spatio-temporal strategy,three Arabidopsis senescence-related genes(At1g69490,At1g66390 and At5g45890)were selected and their promoters were used to drive the expression of the secondary wall core transcription factor VND7.To verify the effect of this method,the expression of the target gene VND7 and secondary wall biosynthesis-related genes was analyzed.At the same time,the cell wall structure and cellulose deposition were investigated.The main results are as following:1.We have constructed vectors in which the promoters of three Arabidopsis senescence-related genes are driving the expression of VND7 gene,We transformed these vectors in Col-0 by agrobacterium-mediated transformation,and screened for positive plants.2.We have performed qRT-PCR of the target gene VND7 and three secondary wall cellulose synthase genes(AtCesA4,AtCesA7 and AtCesA8)in the senescent rosette leaves of transgenic plants.Compared with wild-type Arabidopsis(Col-0),the expression level of VND7 and secondary wall Ces As increased.3.We have done microscopic analysis of transgenic Arabidopsis senescent rosette leaves by Calcofluor staining.Compared with Col-0,we found that there were spiral cellulose deposition in the petiole and veins of Pro.At1g69490-VND7 transgenic line# 2,and block pattern deposition in secondary cell walls of the petiole of Pro.At1g69490-VND7 transgenic line # 15 which indicated secondary wall thickening occurred in Arabidopsis.As a lignocellulosic material,grasses provide a large amount of non-polluting and renewable raw materials for the production of biofuels.However,there is an recalcitrance barrier in the plant cell wall,which makes the utilization of lignocellulose difficult.The CslF6(cellulose synthase-like F 6)gene encodes a mixed-linkage(1-3,1-4)-beta-Dglucan synthase,which is mainly found in grasses.Mixed-linkage(1-3,1-4)-beta-Dglucan is a cell wall matrix polysaccharide that can affect cell wall structure.In this study,we constructed OsCslF6 overexpressing rice material,and then examined the agronomic traits,determined the cell wall composition and analyzed the enzymatic hydrolysis efficiency of cellulose.The results are as follows:1.The expression level of OsCslF6 in OsCslF6 overexpressing rice increased.2.The breaking force of OsCslF6 overexpressing rice increased,and the 1000-grain weight decreased.3.The cell wall hemicellulose content increased in OsCslF6 overexpresses rice stems.4.Under direct enzymatic hydrolysis,the enzymatic hydrolysis efficiency of OsCslF6 overexpressing rice was increased up to 94.03% compared with NPB;Under the condition of 10% CaO pretreatment,the enzymatic hydrolysis efficiency of OsCslF6 overexpressing rice lines was increased up to 22.81% compared with NPB;Under the condition of 200℃ hot water pretreatment,the enzymatic hydrolysis efficiency of OsCslF6 overexpressing rice lines increased 57.05%.