The Construction Of Metagenomic Library From The Alkalescence Environmental Samples And The Clone Of Alkaline Protease Gene AP01

The extremophiles belong to a huge and potential superiority gene resource library and the research of uncultured microorganisms has been very important in the extremophiles research. In the nature soil, it is generally accepted that more than 99% of bacteria and fungi have not been cultivated for study in pure culture. It's an important method to study microbial gene resource from molecular biology with extracting and purifying the metagenomic DNA directly to construct library and it's also the key way to research the diversity and physiology characters of uncultured microorganisms.Now, the method and technology of how to extract and purify the metagenomic DNA from soil samples are still imperfect. There is rare public paper to discuss the methods of how to extract and purify the total DNA from the alkaline soil samples directly. By now, there has been no formal paper to report the study of constructing the alkaline soil library to research the alkaline environmental microbial gene resources publicly,especially the uncultured microorganisms.Alkaline protease is a very important group of enzymes in the physiology and commerce. It has been used widely to the detergents, leather processing, furriery and silk industry. Besides, it has a wonderful prospect of potential application in medicine, food processing, feeds, environment protection and chemical industry, as well as the basic research.In this study, the metagenomic DNA were extracted and purified from alkalescence environmental samples directly from one paper factory of Nanning city in Guangxi Pro.. The crude total DNA yields had been got ranged from 10 to 30 p,g per gram of dry environmental sample. In this method, the metagenomic DNA library which was named AL01 was constructed by inserting restriction fragments of the purified DNAs into plasmids pGEM-3Zf(+) vector and then transferred into DH5a after the genomic DNA which were digested with the restriction enzyme EcoRl partly. AL01 library contained 23,650 positive clones and the average foreign DNA fragments were about 3.2 kb. The length of the library covered 75.68MB. And the efficiency of the metagenomic library was approximately 6,000 clones which contained random inserting foreign DNA fragments from lg dry soil samples.After screening the metagenomic library with the screening tactics of protease activity, we had got a positive clone which code number waspGXAA2011. This clone produced protease to hydrolyze defatted-milk on the special plates. The bio-informatics analysis indicated that this clone foreign DNA fragment contained an integrated ORF which coded 382 amino acids. The GC containing was 46.3%. The foreign DNA fragment contained an integrated alkaline protease gene after analyzed by BLAST software. The protein Mw was 39462.36 D and its pi was 8.91 after predicated by bio-information software EXPASY.The enzyme data had proved the enzyme reaction optimum pH was 9.5 and the optimum temperature was 40 °C when this clone produced alkaline protease.Then we named the protein which coded by foreign DNA fragment AP01 gene and registered it in the Genbank database. The registered number was AY953434.