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Construction of Aquaculture Water Metagenomic Library in Pearl River Delta Area and Chitinase Gene Screening and Analysis

Author: LiHuiQin
Tutor: LinZuoTie
School: South China University of Technology
Course: Microbiology
Keywords: Fosmid library Aquaculture water Chitinase Clone library Denaturing gradient gel electrophoresis
CLC: S917
Type: Master's thesis
Year: 2011
Downloads: 90
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Abstract


Shrimp farming is one of the pillar industries of aquaculture in China. With the constant expansion of shrimp farming, shrimp farming pollution is a growing problem. Shrimp debris accumulation in the culture environment pollution one of the reasons, the main ingredient of shrimp debris chitin. Chitinase major significance for the governance of shrimp farming pollution. Firstly, using denaturing gradient gel electrophoresis (DGGE) and clone library research community structure of the flora of the Pearl River Delta region in shrimp aquaculture water chitin degradation. On this basis, build a shrimp farming the water body macro genome Fosmid library, preliminary access to the shrimp farming the water body's microbial genetic information eventually use functional screening of this library screening to four chitinase quality positive clones, they carried the gene sequence determination, as well as some of the enzymatic function. Significant functional microbes from shrimp ponds in situ environmental identification and screening of potential prebiotic effect. The main results are as follows: a denaturing gradient gel electrophoresis technology used in different breeding period of shrimp farming water chitin degradation flora, fingerprint analysis show: breeding interim shrimp farming water of chitin degradation flora diversity compared with late rich. b. chitinase gene clone library to build 18 family, the study of shrimp farming waters chitin degradation flora communities: shrimp farming water chitin-degrading bacteria, including the genus Bacillus (Bacillus) and uncultured bacteria, accounted for 45% and 40%, respectively. In addition, further comprising: a bacterial heap capsule sticky genus (Sorangium cellulosum), Serratia marcescens (Serratia marcescens), the genus Streptomyces (Streptomyces roseosporus), the genus Arthrobacter (of Arthrobacter SP.) Rich species. And uncultured chitin degrading bacteria are actinomycetes. c. using low melting point agarose embedding extracted metagenomic DNA, water shrimp farming a metagenomic Fosmid library was constructed on this basis, the library received a total of clone 8000 inserted fragments average 35kb (25 to 58Kb) are built library capacity of 280Mb. When the continuous passage 100, the library is still maintain good stability. d. the colloidal chitin screened tablet from 8000 clones screened the four positive clones were named CF1, CF2, CF3, CF4. CF1 up to 94% higher with a nitrate reduction properties of Bacillus cereus sequence homology. The CF2 high sequence homology microbes are uncultured production chitinase microbial gene sequence conservative catalytic fragment of the 18 family. CF3 and sequence of Streptomyces ghanaensis ATCC 14672, 100% homology. preliminary study e. the CF2 clones enzymatic properties of the enzyme optimum pH 7.0, optimum temperature of 35 ° C, the activity reached 0.128U/ml optimal conditions, higher activity.

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CLC: > Agricultural Sciences > Aquaculture, fisheries > Aquatic basic science > Aquatic Biology
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