Construction Of Aquaculture Water Metagenomic Library In Pearl River Delta Area And Chitinase Gene Screening And Analysis

With the rapid development of shrimp farming,the conditions of shrimp water is getting worse.One reason is accumulation of shrimp residual body which mainly composed of chitin. So,studies on chitinase are helpful for solving the pollution of shrimp water. In this paper,we firstly analyzed the community composition of chitin-degrading bacteria by denaturing gradient gel electrophore-si(sDGGE)and clone library basing on chitinase gene. Secondlly,a fosmid library of shrimp water was constructed four clones expressing chitinase activities were screened from the fosmid library. Sequencing analysis and enzyme activity were studied. These result made great sense in screening in-siu functional bacterias of shrimp water.The main results are as follows:a. DGGE was applied in the study of chitin-degrading bacterias in shrimp water sample at different times. DGGE fingerprint analysis revealed that the chitin-degrading bacteria diversity in the middle stage of the culture is higher than the later.b. A clone library based on 18 chitinase gene was constructed to study the community composition of chitin-degrading bacteria. Results showed that chitin-degrading bacterias in shrimp water is mainly consisted of Bacillus and uncultured bactiria which the percentage is 40% and 45% respectively;The other bacteria like Sorangium cellulosum,Serratia marcescens, Streptomyces roseosporus,Arthrobacter sp., were found in the library. The uncultured bacterias belongs to the actinomycete.c. Megabase-sized DNA fragments were prepared by embedding. A fosmid metagenomic library of uncultured microorganism from shrimp pond water was constructed with the capacity of 280Mb in which the average inserted fragement size at about 35kb and totally stored 8000 clones. The fosmid library clones cultured ford. Functional screening was applied to search for the positive clones that expressing chitinase activity. Four positive clones were screened out from the fosmid clone library, which were named as CF1,CF2,CF3,CF4 respectively. Sequencing analys suggested that the CF1 has a high sequence similarity with Bacillus cereus. CF2 has a high sequence similarity with uncultured bacteria. CF3 had high similarity with Streptomyces ghanaensis ATCC 14672.And we found that there has no reports about the function of chitinase with this bacteria.e. Crude enzyme of the CF2 clone was preliminarily characterized. The result showed its optimal PH was 7.0 and the optimal temperature was 35℃.In the optimal conditions,the enzyme activity was 0.128U/ml.