大额牛在我国仅分布于云南，为半野生半家养的珍稀牛种资源，主食竹子，具有粗纤维消化能力强的特点。本项目利用已构建好的瘤胃细菌Fosmid文库（09年国家自然基金），拟筛选具有纤维素酶活性克隆400个，测序，组装；与参考数据库（NCBI的NR、COG、KEGG、STRING、Swiss-Prot及CAZy数据库）Blastx比对,进行物种注释和基因功能分析；用DNAman等基因分析软件分析新纤维素酶基因结构，预测高活性位点；在E.coli表达系统中表达新纤维素酶基因，检测表达产物的酶学性质并根据研究结果采用易错PCR/DNA改组技术对其进行定向改造，以进一步提高其稳定性和/或酶活力；预期揭示大额牛高效利用纤维性饲料和采食竹子的瘤胃微生物基础，筛选高效分解粗纤维的基因和酶，结果可为反刍动物饲料高效转化和生物能源的利用提供一条有效途径，对大额牛的人工饲养及生物资源的开发具有重要的意义。

The gayal is a rare semi-wild bovine species distributed throughout china. They can graze grasses including bamboo as well as reeds and other plant species, and attain higher mature live weights than those of cattle maintained in similar environments. Based on the research of National Natural Science Foundation of China (Grant No.30960256),this project will take advantage of a metagenomic library of circa 348,00 fosmids which have been constructed from bacterial DNA isolated from gayal rumen. We will plan to screen 400 clones for cellulose hydrolysing activities and sequence. All reads will be assembled using SOAPdenovo, with specific parameter for metagenomics data.Taxonomic assignments of all reads will be made on the basis of BLASTx search of the NCBI-NR database. Function analysis of all cellulase genes will be made on the basis of BLASTx search of the NCBI-COG, KEGG, STRING, Swiss-prot and CAZY database. We will try to analysis new cellulase gene structure, to find functional active site, to express new gene in E.coli and detect cellulase biochemical characterisation. Furthermore, based on the results of cellulase biochemical characterisation, directed evolution techniquees of error-prone PCR and DNA shiffling will be used to improve the thermo stability or activity of cellulase. Results from this project will not only provide molecular basic data which ruminal microbe of gayal can more effectively degrade fibrous feed and bamboo, but also provide an effective way for conversion of ruminant feeds, artificial rearing of gayal and development and utilization of bioenergy.