Screening Of Decomposing Bacteria From Rotten Wood And Corn Stalk And Preliminary Analysis Of 1-5 Function Of Transgenic Bacteria

Biomass energy is the solar energy in the form of chemical energy stored in the biomass form of energy,is one of the important energy to the survival of humans and is second only to coal,oil,natural gas.Because of the biodegradability and non-polluting nature of biomass energy,all countries in the world are competing to develop the materials of postgraduates.Up to now,the research on biomass utilization has been used in the screening of wild strains,and few of them have used genetic engineering techniques to reconstruct the strains so as to degrade their biomass.It is well known that both manganese peroxidase and lignin peroxidase are used to induce enzymatic decomposition of lignin,cellulose,or hemicellulose by altering redox status in cells.But no one has tried to constructe vector of plant active oxygen protease and the active oxygen-related gene is transferred into the receptor strain,which forms the engineering strain to degrade the biomass.The aim of this study was to try to construct transgenic strains from plant active oxygen protease construction vectors to the selected suitable wild strains,and to explore the ability of enzymatic degradation of lignin,cellulose and hemicellulose by controlling plant reactive oxygen metabolism.First of all,biomass such as rotten wood and straw were used as experimental materials to screen potential wild strains associated with degradation of cellulose,hemicellulose or lignin by using guaiacol method and phenotypic isolation method in this study.In the process,different kinds of rotten wood were collected from the Eastern campus of Nanyang Normal University,the front garden of the central Shaw Building,the Nanyang Baihe Wetland Park and the Xichuan County in Henan Province,the original forest around the FA Hai Monastery and different kinds of corn stalk also collected from the Zhaoguanzhuang village and the Baita villages in the northern suburbs of Nanyang.The experiment was divided into two groups,a group of wood was used as experimental materials to screen the wild strains related to degradation of lignin by using the method of guaiacol phenol.Another group of corn straw as the experimental material,according to the number of bacteria growing on the rotten corn stalk to isolate the potential degradation of corn stalk of wild strains.3 strains were obtained by the guaiacol method,which could produce a color reaction and a good growth bacterium,which was identified as Bacillus spp.by molecular identification Because there is no readily available technique in the process of producing the sensation and transformation,the transgenic Bacillus strains related to the active oxygen metabolism of Arabidopsis thaliana have not been successful.However,during the screening process of corn stalk degrading bacteria,we obtained 1 strain that was able to grow on corn stalks and was able to reproduce quickly,and selected it as receptor strain,and identified the strain as Erwinia strain by universal primers sequencing and named 1-5 strain.Secondly,the 1-5 strain screened by corn stalk is the receptor strain of the enzyme genes related to Arabidopsis thaliana active oxygen metabolism.Two transgenic 1-5 strains,named 1-5(APX1)and 1-5(SOD1),were successfully constructed by preparing 1-5 receptor and molecular biological techniques.On this basis,the growth curves of them were analyzed and the protein expression of transgenic proteins was determined by crude protein extraction and protein imprinting assay of two transgenic strains.The transgenic strains and the receptor strains were used to extract protein,explore the optimum conditions,and observe the strips after running the protein glue.The protein was then purified to test the strip.However,the protein expression of the strains was smaller because of the method.Finally,the degradation efficiency of the three strains and composite strains strains for lignin,cellulose and hemicellulose were measured by means of the fan washing method,and the degradation efficiency of each group increased with the degradation time.After the records were detected,the transformed single strains were found to be relative to the original strains for lignin,the degradation efficiency of cellulose and hemicellulose was about 5%,and that of 1-5(SOD1)was slightly higher than that of 1-5(APX1),and the compound conversion strain was about 5% higher than that of single conversion strain,about 10% higher than that of wild strains.It is indicated that the transgenic APX1 and SOD1 genes can improve the degradation efficiency of 1-5 strains to corn stalk,and also show that the degradation efficiency of corn stalk under the action of compound strain is relatively higher.