天然红树林土壤微生物大片段宏基因组文库的构建

采用改良及优化后的原位裂解法提取红树林土壤环境DNA，通过脉冲场电泳图谱显示所得DNA片段主要集中于23～50 kbp范围, 表明大片段DNA在总DNA中的比率明显提高; 对改良后的原位裂解提取法与不同DNA纯化方法的组合进行比较分析, 确定电洗脱为最佳纯化方法; 运用改良后的原位裂解提取法与电洗脱纯化法联用实验方案，构建了4个季节天然红树林土壤Cosmid宏基因组文库. 随机挑取克隆子，提取质粒后经BamHI酶切验证, 平均插入片段均大于35 kbp,且片段类型各不相同, 推测该文库至少包含了335 Mbp潮间带微生物基因组信息. 该方法的建立使得开发红树林湿地生态系统的特色微生物资源成为可能, 并为探索其它生境的未培养微生物资源提供借鉴.

Construction of Large Fragment Metagenome Library of Natural Mangrove Soil

Applying our optimized direct extraction method, the percentage of large fragment DNA in the total extracted mangrove soil DNA was significant increased. The large fragment metagenome library derived from natural mangrove soil over four seasons was successfully constructed by the optimized DNA extraction and electro elution purification method. All of the clones had recombinant Cosmids and each differed in their fragment profiles when Cosmid DNA was extracted from 12 randomly picked colonies and digested with BamHI. The average insert size for this library was larger than 35 kbp. This culturing-independent library at least encompassed 335 Mbp valuable genetic information of mangrove soil microbes. It allowed mining of valuable intertidal microbial resource to become a reality. It is a recommended method for those researchers who have still not circumvented the large insert environmental libraries or for those beginning research in this field, so as to avoid them attempting repetitive, fussy work.