Cloning And Expression Of Key Genes For Fungal Biotransformation Of Lignite

Coal bioconversion is an efficient coal utilization technology with mild reaction conditions,low energy consumption,and environmental friendliness.In the more than 40 years since the beginning of research abroad,coal bioconversion technology has developed vigorously with the efforts of researchers at home and abroad.However,there are still some problems in coal bioconversion technology,the most critical of which is the breeding of coal-degrading microorganisms.Inview of the current difficulties encountered in the breeding of coal-degrading microorganisms,this thesis attempts to use genetic engineering technology to transform fungi,provided a new reference for genetic engineering of coal-degrading fungi.The main findings are as follow:(1)The genome information of Trichoderma harzianum published on NCBI website was used for comparison,and then four genes suitable for overexpression were screened by multiple analysis tools and their complete gene sequence was amplified.The amplified genes differ only a few bases from the published genes,and all can be translated into complete proteins that can be used for gene overexpression.(2)Meanwhile in order to understand the lignite degradation characteristics of AH bacteria,solid plate culture and liquid fermentation experiments were carried out.The results showed that the ability of AH bacteria to liquefy lignite was related to the degree of oxidation of lignite.The higher the degree of oxidation,the easier it was to be liquefied;The liquid fermentation experiment found that the addition of nitrifying lignite can increase the weight of AH bacteria hypha by nearly 60%,and AH bacteria can degrade nitrifying lignite and produce a large amount of humic acid,and make the weight loss rate of nitrifying lignite reach 60%,this indicated that AH bacteria could not only secrete some extracellular substances to dissolve pulverized coal,but also secrete extracellular enzymes to degrade pulverized coal particles in the process of culture,and the degraded small molecular substances could promote the growth of mycelia.The results of transcriptomic experiment and qRT-PCR showed that the addition of lignite powder could promote the expression of lignin-degrading enzyme gene in AH bacteria;Infrared spectrum analysis showed that AH bacteria had a strong ability to degrade lignite containing carbonyl functional groups and aromatic compounds with side chains.(3)Transform the existing pkd7-RFP plasmid to obtain pkd7-HYG plasmid,making it more suitable for the transformation of AH bacteria and the selection of transformants,connect the target gene with pkd7-HYG plasmid to obtain recombinant plasmids pkd7-HYG-340,pkd7-HYG-348,pkd7-HYG-463,pkd7-HYG-568.After introducing each plasmid into Agrobacterium AGL-1,the transformation experiment of AH strain was conducted using the modified ATMT method.Each overexpression strain was obtained: 8 AH-340 transformants,8 AH-348 transformants,8 AH-463 transformants,and 12 AH-568 transformants.(4)The genetic stability of each transformant was detected by PCR,and transformant strains with high gene expression and high extracellular protein content were selected for lignite degradation experiments.The results showed that when the overexpressed strains degraded lignite,the weight of mycelium increased in varying degrees compared to the blank group without lignite addition.Among them,the mycelium weight of strains AH-340,AH-463 and AH-568 increased the most,reaching about 40%,25% and 10%,respectively.The original AH strain showed almost no change.The acid precipitation results of the fermentation supernatant showed that the precipitation quality of the overexpressed strains AH-340,AH-348,AH-463 and AH-568 after lignite addition was significantly higher than that of the original AH bacteria.When overexpressing AH strain degrades lignite,it can produce more humic acids than the original AH strain,and the weight of mycelia is also significantly increased,indicating that the overexpression of gene can improve the coal degradation ability of AH strain to some extent.This thesis has 33 figures,12 tables and 95 references.