| Object:Ice and frozen soil resources are very abundant in our country, but compared with the relative domestic and foreign, cold-adapted microorganisms development research is still relatively backward, the domestic research in psychrophilic yeast to near zero. As global warming, glaciers in the degraded with each passing year, so the study of low temperature microorganisms is around the corner, because the yeast is closely related to human life, low temperature yeast research has grown ever more important, according to the current worldwide understanding of the depth and width of the development and utilization of psychrophilic yeast, there is also a serious shortage, This topic is to use Urumqi Tianshan mountain glacier NO.1separate screening psychrophilic yeast resources, research and its genetic diversity and molecular phylogeny, further understand the status of the psychrophilic yeast of ecological, physiological, biochemical, and thus can filter out a great deal of adaptive low temperature strain of the ecological environment, so as to better understanding of cold-adapted microorganisms, and system development situation.Believe that along with the advance of science and technology around the world and communicate with each other to increase, the study of low temperature microorganisms and their related products in China will achieve certain results.Methods:The NO.1of Tianshan mountain glaciers in Urumqi river head is a glacier hydrology, climate change and ecological basic research aspects, such as the most detailed glaciers in our country, this study based on unique this cryogenic geographical environment in Xinjiang, from empty water and sediments at the bottom of the glacier melt water of the NO.1of Tianshan mountain separation in the screening of psychrophilic strains of yeast, the strains of isolation and purification of medium and purification medium to have separate strains of the optimum growth temperature, the optimum pH and salt resistance test, through pure culture method separating Tianshan mountain glaciers psychrophilic yeast diversity and sequencing studies the diversity system, ITS and26S rRNA gene sequence analysis is used to determine the system of psychrophilic yeast species evolution status, using molecular biology method to reveal the species diversity and phylogenetic relationship of strain to understand ecological physiology; Using macro genomics technology of Tianshan mountain glacier water samples collected in the establish of Tianshan mountain glacier cold-adapted microorganisms gene library, in order to get a lot from not cultivate microbial genetic information; from the pure culture screening, purification low-temperature enzyme production strains of yeast, including esterase, lipase and amylase, protease, pectinase and chitinase, through selective medium were tested strains produce extracellular enzyme properties, and through the MSP-PCR fingerprint technology to26S rRNA genes to further distinguish between highly homologous strains, strain characteristics of extracellular enzyme production is studied, in order to further research and development of psychrophilic yeast of biotechnology used provides the basis, and intends to solve the problem of low temperature enzyme source, particularly with regard to their ability to develop as a source of cold active enzyme and biological degradation ability of study.Results:1Through dilution plate a total of66strains were isolated from culture, the indeed for yeast were determined under the microscope, and most isolated strains can produce pigment (68.4%), colony color is bright red, orange, yellow fraction, milky white, such as separation of cold-adapted microorganisms, and the optimum growth temperature of18to24℃, the vast majority belongs to cold resistant bacteria. After determination of26S rDNA and ITS sequence analysis, analysis of system development, the low temperature can foster points belong to basidiomycotina strains in the development of the tree (basidiomycetes), ascomycetes classes, type of yeast (yeast-like organisms)3system development groups, including basidiomycotina as the advantage bacterium group. Cryptococcus genera is the dominant strains genera, accounts for the proportion of the total number of strains, the largest glaciers in the largest contribution to the culturable microbial diversity in low temperature, the proportion of enzyme production most, followed by red saccharomyces (Rhodotorula) and throwing spore saccharomyces (Sporobolomyces) and wood, saccharomyces (Mrakia).This study suggests that Tianshan mountain glaciers is preservation of the natural production, it can cultivate cold-adapted microorganisms has rich diversity.2Macro genomics technology was used to construct Tianshan mountain glaciers fungal gene clone library at low temperature, positive clones tested by microbial PCR, after double enzyme gene sequencing getting94sequence, including14basidiomycete (Basidiomycota), ascomycetes (Ascomycota), a total of47,1chytrid fungus(Chytridiomycota),32unknown fungi(uncultured fungus).Ascomycota is the largest library in ascomycetes the evolution of clusters, and paecilomyces (Simplicillium sp.) zand Aspergillus (Aspergillus sp.) for a larger proportion,17.02%and27.66%, respectively, the unknown fungi (uncultured fungus) in the second, divided into false powder capsule bacteria genera (uncultured Pseudeurotium) and false basidiomycete fungi genera (uncultured Basidiomycota).Studies have shown that molecular methods than artificial cultivation can gain more genetic information, more rich diversity, and can more accurately reflect the Tianshan mountain glaciers fungal distribution in the low temperature microorganisms.3This study through to have separate screening yeast, a total of47of low temperature enzyme production strain, including15strains can produce lipase,21strains can produce amylase,12strains can produce pectinase,17strains can produce protease,5strains produced chitinase in low temperature. Most of the enzyme production strains obtained the optimum growth temperature is about20℃(at room temperature), the growth temperature range between4℃-24℃, basic belongs to the category of cold resistant strains. Optimum growth in7%NaCl concentration, most bacteria belong to the micro well salt,47of low temperature enzyme production strain respectively belong to basidiomycotina, ascomycetes classes, type of yeast (yeast-like organisms)3system development groups, including basidiomycotina as the main strains enzyme production. The experimental conclusion shows that the yield of psychrophilic yeast and low temperature enzyme ability big and rich, therefore, in the Tianshan mountain glacier contains extremely rich variety of low temperature enzyme production of microbial resources, is the ideal enzyme source. |
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