蔗糖富集环境土壤微生物宏基因组分析及蔗糖水解相关酶基因克隆

Metagenomic Analysis of Microbes from Sucrose-enriched Environment and Cloning of Sucrose Hydrolase Genes

蔗糖水解酶是蔗糖转化生成生物质能源的关键酶,且还具有重要的转糖苷功能.针对蔗糖富集的土壤环境,利用未培养的宏基因组技术对蔗糖水解相关的酶基因进行克隆.首先使用微生态分子技术对蔗糖富集的土壤样品进行分析,在可信区间为95%的情况下,样品覆盖率为20%(C指数为0.2),Species richness指数为235.0,Shannon index为5.2889,说明这个蔗糖富集样品中的微生物来源具有广泛性.然后使用宏基因组技术构建这个土壤样品中微生物的DNA文库,成功构建一个包含约100000个克隆的大片段DNA Fosmid文库.对文库中的Fosmid质粒进行随机测序,发现质粒的外源DNA与已报道的DNA都没有同源性,文库所克隆的DNA都来源于仍没有被研究的微生物.使用蔗糖作为唯一碳源对文库进行筛选,获得了能水解蔗糖的克隆.在蔗糖水解能力最强的两个克隆中所包含的蔗糖水解酶与GenBank数据库中已知蔗糖酶的相似性分别为38%和68%.

The hydrolases for sucrose are the key enzyme in the process of converting sucrose into bioenergy,and they also possess important transglycosylation activity. The genes encoding the sucrose hydrolysis-related enzymes from sucrose-enriched environment were cloned by employing metagenomic techniques. First,micro-ecosystem techniques were used to analyze the samples from sucrose-enriched soil. When confidence interval was 95%,the sample coverage was 20%（C index is 0.2） ,the species richness index 235.0,and Shannon index 5.288 9. These evidences showed that the microbes in the soil sample were diverse. Then,a metagenomic DNA library containing 100 000 clones was successfully constructed. The result from random sequencing of Fosmid plasmids showed that the DNA in the library came from unknown microbes. Positive clones were isolated from the library cultured on the minimal medium plates with sucrose as the sole carbon source. Two clones with the biggest activities to sucrose were analyzed,and the plasmids in the two clones were sub-cloned and sequenced. The genes of sucrose hydrolases in these plasmids showed only 38% and 68% identities with known protein sequences in GenBank.