The Screening Of Cellulase Gene Based On The Metagenomics From High Temperature Daqu And Microbes Structure Analysis

Daqu is used the grain which contains abundant protein as the main raw material, with open starter propagation process, between60～65℃of starter-propagation temperature produced a variety of thermophilic bacillus and protease,lipase,amylase, cellulase,etc, and the unique community.The part1of this thesis:use DaQu as materials, based on the metagenomics method,make extraction of DaQu metagenomics total DNA link Fosmid carrier, construct a metagenomics library which contains10000clones.Getting two strains contains cellulose enzyme gene in screening clones, after the shake flask liquid fermentation was taked to determine the clone that enzyme activity is higer, named for the G1, the G1enzyme preliminary analysis was taked. Connect the G1plasmid enzyme after digested by BanmHI to pUC18-T carrier, constructing the sub-clone library, through the further, screening,the cellulose enzyme gene segment was narrowed down to1169bp or so. sequenced the fragments gene, the analysis shows that the DNA sequences have100%of the similarity with bacillus licheniformis (GenBank registration number CP000002.3); The nucleic acid sequence have a ORF contains147nucleotides, according to the glycosyl hydrolysis enzyme naming rules, the gene was named umcellH, umcellH encoded proteins that molecular weight is expected to size of5243.4Da, isoelectric point is11.3, the1～22amino acid is possible signal peptide.The part2of this thesis:use ribosomes amplification clips extinction enzymes Analysis (Amplifed Ribosomal DNA Restriction Analysis, ARDRA) technology and Single Conformation polymorphism Analysis (Single-Strand Conformation Polymor-phism, SSCP) technology to the daqu microbes structure Analysis. Through the ARDRA technical analysis of the structure of daqu microbes and the establishment of two16S rDNA librarys, the culture-dependent method library of90clones have23kinds of OTUs, the culture-independent method library of96clones get34kinds of OTUs.In the standard library diversity index, the diversity index of the culture-independent method is Shannon-Wienner(H):4.94; Simpson(D):0.998; Richness(R):28.91; and the index of the culture-dependent method is H:3.11; D:0.077; R:7.23. The diversity index of culture-independent method is higher than the culture-dependent method (3.02,0.062,4.89),the Evenness index(E) of two method are0.88. Each index of the two librarys analysis shows that the culture-independent method reveals the better diversity of DaQu microbes. Using SSCP technology research the microbial community structure of daqu surface, side, center three different parts, through the extracted total DNA from different parts,the PCR amplification of the16S rDNA V4-V5area,amplified products for enzyme cut,degeneration,get the single DNA in non-denaturing polyacrylamide gradient gel electrophoresis, the results showed that the microorganisms of center is most abundant.