Hydrolysis Pretreatment Evaluation Of Transgenic Brachypodium Distachyon Expressing ?-1,4-Endoglucanase

In recent years,people desire for renewable energy along with the shortage of non-renewable energy resources.This trend has been urging researchers to find alternative resources.Plant biomass has been used as renewable energy resource for thousands of years.Recently,more and more scientists are interested in the biofuel.The chemical method was mainly used in the pretreatment of biomass materials.However,high-cost,high energy consumption and a series of environmental pollution become enormous difficulties for chemical treatment.Recently,in biomass ener,based on enzymatic hydrolysis of cellulose has been rapidly developed.Therefore,the mainly purpose of this study is to express ?-1,4-endoglucanase(El)through Agrobacterium mediated transformation in the Brachypodium distachyon(Bd21).Hydrolysis pretreatment evaluation was used for transgenic Bd21.The main results are as follows:1.In the experiment,Agrobacterium mediated transformation system was determined with high stable and efficiently.The best time of embryogenic callus in Bd21 was about 15 d.At this time,the regeneration rate reached the highest(74.89%).The conditions were the best with the infection time of Agrobacterium(0D600=0.5)of 6-9 min,250 mg/L of the hygromycin concentration,and two screening.2.At total 35 putative transgenic plants were obtained from callus regeneration.Three positive plants were identified by PCR and Southern blotting analysis.Western blotting analysis indicated that El gene was expressed in the three transgenic lines and also expressed in stems,leaves and seeds of transgenic lines.E1 protein was not detected in the root and wild-type Bd21.3.The enzyme activity of E1 was detected in three transgenic plants.The enzyme activity of cellulase El was highest in transgenic plant 2,reaching 15000 MU/mg protein/min pmol.The activity of cellulase El in different organs also showed different activities.The enzymatic activity of cellulase E1 in leaves was the largest,followed by the activity of cellulase E1 in the stem,seed and root.The wild-type plants exhibited no E1 activity.4.According to the response surface analysis,the optimum process conditions were as follows:30.00 g/L,4.34,54.76 ? 72.00 mesh and 50.00 h of substrate concentration,pH,temperature,particle size and time,respectively.Finally,the yield of reducing sugar was 35.891 + 0.67%in the transgenic Bd21(theoretical prediction of 38.3123%,the control group of 12.927%).