利用宏基因组文库筛选草甘膦不敏感的5-烯醇式丙酮莽草酰-3-磷酸合酶（EPSPS）基因

Screening glyphosate insensitive 5-enolpyruvyl shikimate-3-phosphate synthase （EPSPS） gene from metagenomics

为探索利用宏基因组文库筛选草甘膦不敏感的5-烯醇式丙酮莽革酰-3-磷酸合酶（EPSPS）基因，直接从土壤中提取细菌DNA，构建草甘膦污染土壤细菌宏基因组文库，并利用EPSP合酶缺失突变株ER2799筛选到4个能互补EPSP合酶功能的克隆，其中两个克隆的转化子能够在含5mmol／L草甘膦的MOPS培养基上生长。测序结果表明它们均含有完整的EPSP合酶编码基因，GT-1aroA核苷酸长度为1335bp，编码445个氨基酸，GT-4aroA核苷酸长度为1350bp，编码450个氨基酸。利用PCR方法将两个基因克隆到原核表达载体pET28a上后，可以使宿主细胞BL21（DE3）在含100mmol／L草甘膦的MOPS培养基中良好生长。上述结果表明，利用宏基因组文库筛选方法可以得到高抗草甘膦的EPSPS基因。

To explore the feasibility of applying metagenomic libraries to obtain the glyphosate insensitive 5-enolpyruvyl shikimate-3-phosphate synthase genes, metagenomic library of soil which was extremely contaminated with glyphosate was constructed by directly isolating DNA, and 4 clones which can complement EPSPS＇ function were screened by using aroA - mutant E. coil ER2799. Two of them can grow on MOPS media supplied with 5mmol/L glyphosate, GT-1aroA has 1335bp fragment encoding 445 amino acids and GT-4aroA has 1350bp fragment encoding 450 amino acids. Two aroA genes were cloned into pET28a vector by PCR and transformed into E. coli BL21 （DE3）, both of transformants showed glyphosate insensitive and growed well on MOPS media supplied with 100mmol/L glyphosate. The results showed that metagenomic libraries screening method could be applied to obtain the new glyphosate insensitive 5-enolpyruvyl shikimate-3-phosphate synthase genes in future.