Jiang Jian-Dong, Gu Li-Feng, Sun Ji-Quan, Dai Xian-Zhu, Wen Yang, Li Shun-Peng
College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural Environment of Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China.
Sheng Wu Gong Cheng Xue Bao. 2005 Nov;21(6):884-91.
Construction of multifunctional pesticides-degrading genetically engineered microorganisms (GEMs) is increasing important in the bioremediation of various pesticides contaminants in environment. However, construction of genetically stable GEMs without any exogenous antibiotic resistance is thought to be one of the bottlenecks in GEMs construction. In this article, homologous recombination vectors with the recipient's 16S rDNA as homologous recombination directing sequence (HRDS) and sacB gene as double crossover recombinants positive selective marker were firstly constructed. The methyl parathion hydroalse gene (mpd) was inserted into the 16S rDNA site of the carbofuran degrading strain Sphingomonas sp. CDS-1 by homologous recombination single crossover in the level of about 3.7 x 10-(7) - 6.8 x 10(-7). Multifunctional pesticides-degrading GEMs with one or two mpd genes inserted into the chromosome without any antibiotic marker were successfully constructed. The homologous recombination events were confirmed by PCR and southern blot methods. The obtained GEMs were genetically stable and could degrade methyl parathion and carbofuran simultaneously. The insertion of mpd gene into rrn site did not have any significant effect on recipient' s physiological and original degrading characteristics. The methyl parathion hydrolase (MPH) was expressed at a relatively high level in the recombinants and the recombinant MPH specific activity in cell lysate was higher than that of original bacterium (DLL-1) in every growth phase tested. The highest recombinant MPH specific activity was 6.22 mu/tg. In this article, we describe a first attempt to use rRNA-encoding regions of Sphingomonas strains as target site for expression of exogenous MPH, and constructed multifunctional pesticides degrading GEMs, which are genetically stable and promising for developing bioremediation strategies for the decontamination of pesticides polluted soils.
构建多功能农药降解基因工程菌（GEMs）在环境中各种农药污染物的生物修复中变得越来越重要。然而，构建没有任何外源抗生素抗性的基因稳定的GEMs被认为是GEMs构建中的瓶颈之一。在本文中，首先构建了以受体菌的16S rDNA作为同源重组导向序列（HRDS）和sacB基因作为双交换重组体阳性选择标记的同源重组载体。通过同源重组单交换，将甲基对硫磷水解酶基因（mpd）以约3.7×10⁻⁷ - 6.8×10⁻⁷的水平插入到呋喃丹降解菌株鞘氨醇单胞菌属（Sphingomonas sp.）CDS - 1的16S rDNA位点。成功构建了将一个或两个mpd基因插入染色体且无任何抗生素标记的多功能农药降解GEMs。通过PCR和Southern杂交方法确认了同源重组事件。所获得的GEMs基因稳定，能够同时降解甲基对硫磷和呋喃丹。将mpd基因插入rrn位点对受体菌的生理和原始降解特性没有任何显著影响。甲基对硫磷水解酶（MPH）在重组体中以相对较高的水平表达，并且在测试的每个生长阶段，细胞裂解物中重组MPH的比活性均高于原始菌株（DLL - 1）。重组MPH的最高比活性为6.22 μ/tg。在本文中，我们首次尝试将鞘氨醇单胞菌属菌株的rRNA编码区域用作外源MPH表达的靶位点，并构建了基因稳定且有望用于开发农药污染土壤去污生物修复策略的多功能农药降解GEMs。
