摘要
构建遗传稳定的多功能农药降解基因工程菌可以为农药污染的生物修复提供良好的菌种资源,然而,构建遗传稳定且不带入外源抗性的基因工程菌是一个难点。通过以受体菌的16SrDNA为同源重组指导序列、sacB基因为双交换正筛选标记构建同源重组载体,二亲结合的方法将甲基对硫磷水解酶基因(mpd)整合到呋喃丹降解菌Sphingomonassp.CDS1染色体的16SrDNA位点,分别成功构建了含1个和2个mpd基因插入到rDNA位点且不带入外源抗性的基因工程菌株CDSmpd和CDS2mpd。同源重组单交换的效率为3.7×10-7～6.8×10-7。通过PCR和Southern杂交的方法验证了同源重组事件。基因工程菌遗传稳定,能同时降解甲基对硫磷和呋喃丹。甲基对硫磷水解酶(MPH)的比活在各生长时期均高于原始出发菌株,比活最高达6.22muμg。
Abstract Construction of muhifunctional 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 the16S rDNA site of the carbofuran degrading strain Sphingomonas sp. CDS-1 by homologous recombination single crossover in the level of about 3.7×10^-7-6.8×10^-7. Muhifunctional 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/μg. 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.
出处
《生物工程学报》
CAS
CSCD
北大核心
2005年第6期884-891,共8页
Chinese Journal of Biotechnology
基金
国家"十五"科技攻关重点项目(No.2004BA516A02)
国家高技术发展计划"863"(No.2004AA246070
2004AA21410)
江苏省研究生创新计划项目资助~~
关键词
同源重组
RDNA
sacB
基因工程菌
农药降解
生物修复
homologous recombination, rDNA, sacB, genetically engineered microorganisms, pesticides degradation,bioremediation
