天山一号冰川底部沉积层产蛋白酶耐低温菌株的筛选及其系统发育

Phylogenetic and physiological diversity of cold-adapted bacteria producing protease from sediments of the bottom layer of the Glacier No． 1 in the Tianshan Mountains

摘要:

摘要:【目的】通过对天山一号冰川底部沉积层耐低温菌的分离和其中产蛋白酶菌株的筛选，了解冰川微生物生理多样性和系统发育多样性，为高效低温蛋白酶生物技术的研发奠定基础。【方法】采用稀浓度的R2A、TSB平板涂布分离可培养细菌，通过脱脂乳选择性培养基筛选产蛋白酶的耐低温菌株。对分离菌株表型特征、生理生化特性、最适生长温度、耐盐性、产酶性能进行了比较，结合16S rRNA基因序列同源性分析确定产蛋白酶菌株的多样性和系统进化地位，通过BOX-PCR指纹技术分析16S rRNA基因序列高相似度的近缘菌株的遗传差异。【结果】从125株分离物中筛选到27 株产蛋白酶的耐低温菌株，其中21株为适冷菌，仅6株菌为专性嗜冷菌，革兰氏阴性菌居多，假单胞菌属( Pseudomonas)菌株占40.7%。产酶菌株隶属于5个系统发育类群、9个属，其中γ-Proteobacteria、Actinobacteria、CFB(Cytophaga-Flexibacter-Bacteroides)为优势 类群。【结论】天山1号冰川底部沉积层冻土中产蛋白酶的耐低温细菌多样性较丰富，本研究筛选得到的同属近缘种群较多，其产酶性状存在差异，适合开展微生物种群的生物地理学研究。

Abstract:

Abstract:［Objective］We characterized proteolytic bacteria isolates from sediments of the bottom layer of the Glacier No．1 in the Tianshan Mountains，China．Physiological test and phylogenetic analysis were undertaken to expand our knowledge on diversity and ecological distribution of psycrotrophic and psycrophlic bacteria populations．［Methods］Using the screening media containing skim milk，we screened cold-adapted strains producing protease． Taxonomic identity and genetic variability of strains isolated was determined by partial 16S rRNA gene sequences and repetitive-element PCR fingerprint．［Results］Of the total 125 cold-adapted bacterial isolates，high levels of protease activity were observed from 27 isolates at optimal growth temperatures ranging from 15 to 24℃ in plate assay． Among 27 protease-producing strains， only 6 isolates were psychrophilic．The 16S rRNA gene phylogenetic analysis revealed that protease-producing isolates belonged to 5 phyum，namely α、，β and !of Proteobacteria，Actinobacteria and Cytophaga-Flexibacter-Bacteroides． They are affiliated to the genera Pseudomonas， Polaromonas，Brevundimonas， Rholococces，Cryobacterium， Kocuria，Arthrobacter，Chryseobacterium and Flavobacterium．The populations of the predominant cultivated protease-producing bacteria are the Pseudomonas spp．(40.7%)．［Conclusion］The results enriched our knowledge on the phylogenetic and physiological diversity of cold-adapted strains producing protease in cold environments．