农业生物技术学报2012，Vol.20Issue(7)：831-836,6.DOI:10.3969/j.issn.1674-7968.2012.07.014
瘤胃微生物Fosmid文库蛋白酶活性克隆的筛选与序列分析
Screening and Sequence Analysis of Protease Clones from Ruminal Microbial Fosmid Library
摘要
Abstract
During the degrading of diet protein, protease is the first key enzyme which hydrolyses protein to peptide or amino acid. However there is few genetic information of protease from rumen microbiota due to the limitation of pure-cultured method. The objective of the experiment was to screen, sequence and bioinformatics analyze the protease clone from a Fosmid library of the dairy cow rumen microbiota. Using two different protease selective medium, including 1% skim milk or 1% isolated soybean protein, respectively, fourteen protease activity clones were obtained from rumen microbiota Fosmid library containing 30 000 clones. Different protease decomposition ability of the fourteen clones was measured by Folin-phenol reagent method. The results showed that each clone had its unique ability of protease decomposition. When casein was used as the substrate, the range of enzyme activity was from 0.59 to 2.74 U/mg. While the isolated soybean protein served as substrate, the range of enzyme activity was from 0.70 to 7.19 U/mg. Furthermore, the same clone had different sizes of enzyme activity for different substrates. After end sequences often positive cloning(GenBank Accession numbers: JY084410-JY084429), the sequences were blasted by Blastn and Blastx. The results showed that 45% of the genetic sequences could not match with the known genes encoding. The end sequence of prolOF could match to metal peptidase, with the similarity of 54%, which belonged to peptidase family M13. The optimal pH of the clone was 7.0.These results provide the basicdatum for the further study on the clone's enzymatic properties and gene characteristics.关键词
瘤胃微生物/元基因组学/Fosmid文库/蛋白酶/末端序列Key words
Ruminal microbes/ Metagenomic/ Fosmid library/ Protease/ End sequence引用本文复制引用
赵静雯,王加启,赵圣国,卜登攀,周凌云,孙鹏..瘤胃微生物Fosmid文库蛋白酶活性克隆的筛选与序列分析[J].农业生物技术学报,2012,20(7):831-836,6.基金项目
国家重点基础研究发展规划(973)项目(No.2011CB100804) （973）
