The Construction Of Metagenomic DNA Library From The Hypersaline Environmental Samples And The Clone Of A New Amylase Gene

The microbial communities in hypersaline environment is an important resource, it is estimated that >99% of microorganisms observable in nature typically cannot be cultivated by standard techniques. Traditional methods for culturing microorganisms limit analysis to those that grow under loboratory conditions,but useing unculture microorganism technology to molecular analyses of natural microbial communities can resolve this problem perfectly.Construct metagenomic DNA library is proved to be the only best way to study and exploit the genetic diverisity of hypersaline environments.In our research,we used the hypersaline sample as the investigate object.Compare of the published methods for extract and purify of nucleic acids from environmental sample,we developed an high effective method for isolate and purify large fragments of DNA from hypersaline environment,and the DNA after purification can be digested with different restriction enzymes. We used pLAFR3 vector to construct library of the genomic DNA.Twelve positive clones expressing amylase activity were isolated from the metagenomic DNA library. The serial number of L14 positive clone which expressing the highest amylase activity was chosen for further study. The amylase gene (amyl4) was sequenced with the strategy of primer walking. GenBank database searching shows there are no obviously identity of the sequence of amyl4, but the product of amyl4 gene has highest identity(68%) with O-sialoglycoprotein endopeptidase.For further investigated the Amy 14, its enzyme properties was characterized. The optimum terperature of Amyl4 is 70℃ and its optimum value is 8.0.The best concentration of Ca~2+and Na~+ is lmol/L and l-2mol/L for each.And there are also show that Na~+ is absolutely necessarily to the enzyme activity of Amy 14. There have no evidence show highconcentration of Na+ can inhibited the enzyme activity obviously, but high concentration ofCa2+ in reverse.