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Cloning and Expression of Celluase Gene exgl from Lentinula Endodes

Author: WuLinZhi
Tutor: WangJingXue
School: Shanxi University
Course: Cell Biology
Keywords: Cellulase Cloning Expression exg1 gene
CLC: S646.12
Type: Master's thesis
Year: 2011
Downloads: 9
Quote: 0
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Abstract


Our country is a very tight national feed resources, land-less population, the contradiction between human and animal food is very outstanding. To keep our country forage industry and the sustainable development of animal husbandry fodder question, we must resolve the feed problem, otherwise would severely restrict its development. Cellulose is the main plant photosynthesis product, which polymerized from 800-1200 glucose molecules. Cellulose is the most cheap, the most abundant renewable resources. Plant generate as much as 1,500 tonnes of dry matter around the world each year, of which the total amount of cellulose and hemicellulose are 850 million tons, but are rarely used. Therefore, it is particularly urgent and important to development successfully of this potential feed resources. Cellulase can generate cellulose, which is a kind of complex compounds, usually divided into endo-1,4-β-D-glucanase,exo-1,4-β-D-glucannase andβ-1,4- glucosidase. In·recent years, with the rapid development of molecular biology, it is a research focus that constructing the efficient production of gene engineering bacterium through genetic engineering.In this paper, exgl gene sequence from Lentinula edodes was amplified by the polymerase chain reaction(PCR) and reverse transcript polymerase chain reaction(RT-PCR) technology and sequence analysis was made. The prokaryotic expression vector was constructed and transformed into Escherichia coli BL21. The expressed product was analyzed by SDS-PAGE and the cellulase activity was determined by CMC-Na. They are as follows:1. Exgl gene sequence from Lentinula edodes was amplified by the reverse transcript polymerase chain reaction(RT-PCR) technology.2. Utilizing the primitive vector pET-28a to construct prokaryotic expression vector pET-28a-exgl. Sequence analysis indicated that the cloned exgl gene was inserted the correct reading frame of expression vector.3. The recombinant strain containing the expression vectors pET-28a-exgl was induced to express through IPTG. SDS-PAGE analysis indicated that the expressed protein molecular weight was 57kDa. The result show the expressed protein was up to 94.9% with the glycosyl hydrolase family 5 and was energetic by CMC-Na determined. The result also showed that the best induced time of the expression vector pET-28a-exgl was three hour.4. Utilizing the eukarya vector pPIC9K to construct eukarya expression vector pPIC9K -exgl.

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