云南腾冲热泉土壤微生物基因组文库的构建与分析

采用冻融、蛋白酶K、SDS-高盐-加热处理法联合的方法,直接从云南腾冲地区的一个弱碱性高温热泉沉积样品中提取和分离环境混合基因组DNA,产量为每克样品1~2μg DNA,用Promega试剂盒纯化后进行PstⅠ部分酶切处理,电泳回收3~8kb的片段后,构建了pSK( )为载体的基因组文库,共获得25000个阳性克隆,平均插入片段长度为4.6kb。通过随机DNA序列测定和基因注释,发现外源插入片段含有未见报道的序列。

Construction and analysis of a metagenomic library from Tengchong hot spring soil in Yunnan Province

By a combination of freezing/thawing/ proteinase K-based method and SDS/high-salt/heating treatment, the mixed environmental genomic DNA was isolated directly from a hot spring soil in Tengchong, Yunnan, China. With this method, The DNA yield was up to 1 - 2 microg/g soil. After purification with the Wizard DNA clean up system (Promega, Madison, Wis), the mixed genomic DNA was partially digested with restriction enzyme Pst I. Digested DNA fragments of 3 - 8 kb were recovered from agrose gel and ligated to the pSK (+) vector. The ligation mixture was transformed into DH10B strain, resulting in the construction of a metagenomic library with about 2.5 x 10(4) clones. Restriction enzyme analysis revealed that the average insert is about 4.6 kb. Some novel sequences were identified via sequencing and gene annotation analysis of 30 clones randomly chosen from this library.