Study On Cloning And Expression Of Trichoderma Reesei Cellulase Genes In Pichia Pastoris

35%～50% of the total plant dry weight was cellulose. It was the most widely distributed, themost abundant carbohydrates on Earth. For mankind, it was also the largest and most renewablenatural substances. Its degradation was the central link in natural carbon cycle. For the use andconversion of cellulose to solve the world energy crisis, food shortages, pollution problems was ofgreat significance.Cellulase was a kind of enzyme can degrade cellulose. On one hand it can put hydrolysis ofcellulose into glucose and other active ingredients, the other hand it can be enhanced the extractionrate by increasing the permeability of cell wall of plant material (protein, fat, Starch), so cellulasecan be applied to all plants as raw materials industry. However, the natural microbial cellulaseproduction, there are still many problems, for example: low-yield, low activity, product feedbackinhibition by glucose. These issues a great impact on the application of cellulose. Moreover, despitethe generally accepted theory synergies cellulase decomposition of cellulose, but there were manyproblems that need to be studied mechanism.Methanol yeast expression system was a eukaryotic expression system developed in the pastten years. Pichia pastoris as a methanol yeast, had the incomparable advantages than traditionalE.coli expression system and the first generation of yeast expression system-Saccharomycescerevisiae. It had many advantages, such as strong ability to express foreign genes, high stability,available for the post-translational processing of proteins, secretion in the intracellular expressioncan also be expressed, and can be high-density fermentation, particularly suitable for eukaryoticgene expression, had become a very important expression system.In this study, Trichoderma reesei QM9414 as the gene donor to cloning cellulase enzymesystem endo-1,4-β-D,glucanase (EG). exo-1,4-β-D,glucanase (CBH) andβ-1,4- glucanase (BG)gene, its yeast expression vectors were constructed respectively, and transformation into Pichiapastoris by electroporation, induce the expression of cellulose, select works bacteria for highlycellulase gene expression level. Lay the foundation for the further study on the synergies ofcellulose and molecular enzyme engineering.The main findings are:Induced expression of cellulase by sodium carboxymethyl cellulose, the best induced time wasinduced 36 hours. Cloning EGⅡ, EGⅣ, EGⅤand CBHⅡgene of multi-glucanase enzymes system by RT-PCR.CBHⅡgene had been sequenced, the sequences had similarity of 99% with No. M16190.1 inGeneBank, only three different bases and three differences in protein sequence. EGⅡsequences hadsimilarity of 99% with No. MI9373.1 in GeneBank, only five different bases and two differences inprotein sequence.. EGⅤsequences had similarity of 99% with No. Z33381.1 in GeneBank, onlytwo different bases and two differences in protein sequence..Trichoderma reesei DNA as the template to cloning BGLⅠgene by using exon splicing.BGLⅠgene had been sequenced, the sequences had similarity of 99% with No. U09550.1 inGeneBank, only four different bases and three differences in protein sequence..A yeast expression vector pPIC9-CBH were constructed. Single linear vector digested with BglⅡ, transformed into Pichia pastoris by electroporation after recovered, got three transformants andby PCR amplification of which two were for the conversion of CBHⅡgene in Pichia pastoris.