【作者】 张国顺；

【导师】 李顺鹏；

【作者基本信息】 南京农业大学 ， 微生物学， 2005， 硕士

【摘要】 环境污染是困绕世界的一个难题，而微生物修复具有其它修复方法不可比拟的优势，成为当今世界研究的热点。但目前在降解微生物的筛选方面存在一些问题，如获得富集液后很难获得纯培养，获得的纯培养可能不是自然环境的优势微生物等而阻碍了其在实际中的应用；在基因克隆方面，由于基因都是来自纯培养，因此克隆的基因比较单一。而富集液降解能力强，所含的微生物为混合菌群，基因具有多样性，更能适应环境，因此如何开发利用富集液是一个值得研究和探讨的问题。本文从富集液的角度进行了六六六微生物降解方面的相关研究，拓宽了微生物降解有毒化合物的研究范围。 一、采集污染土样，利用传统的富集方法获得了一降解富集液，该富集液在实验室条件下(30℃，180r／min，5％接种量)36小时内能够完全降解5ppm的六六六的四种异构体，降解能力稍有差异。为了开拓生物修复的新途径，采用发酵罐对富集液进行了扩大培养，结果表明富集液和纯培养一样可以扩大培养：30℃下，获得种子液(转速300r／min，通气量6L／min)需要28小时，发酵液(转速300r／min，通气量60L／min)需要20小时，其降解效果与摇瓶富集液相当。 二、采用两种方法提取了污染土壤和富集液的总DNA，并做了适当纯化，根据文献报道的六六六降解基因设计引物，以富集液总DNA为模板成功克隆到了六六六降解相关的五个直接基因：linN、linB1、linC1、linD1、linE1，回收纯化的各lin基因的PCR产物分别与pMD18-T Vector相连，转化E．coli DH5α，获得了各重组质粒的阳性克隆：pLinN，pLinB1，pLinC1，pLinD1，pLinE1。将获得的基因与GeneBank上六六六降解相关的基因作了比较，发现与国际上报道的相关lin基因均具有98％以上的同源性，表明六六六降解方面的基因很保守。根据获得的基因可以推断出富集液中至少有两种以上的纯培养。从污染土壤的总DNA中未能扩增到相关基因，可能是目标微生物含量极低和提取中的损失所致，同时也表明富集更有利于功能基因的获得。 三、为了进一步利用获得的基因进行降解酶的生产和应用，对脱氯酶基因(linN)在大肠杆菌中的表达进行了研究。重新设计引物，引物两端分别添加NdeⅠ和HindⅢ酶切位点，通过PCR方法从pLinN中重新扩增了脱氯酶基因，将其用限制性核酸内切酶Nde Ⅰ和HindⅢ双酶切纯化后与经过同样双酶切处理回收的质粒pET29a酶连，获得重组表达质粒pET29a／LinN，然后转化至E．coli BL21，从而获得表达菌株E．coli更多还原

【Abstract】 Environmental pollution is a worldwide problem. Bioremediation is a good way and has more merits. So it becomes the focus of researchers, but there are some questions before the isolation of degrading bacteria. It is hard to find them from enrichment and some of them have no competitive ability in our environment. The genes cloned from pure culture are single for the source of gene is single. The degrading ability of enrichment including all kinds microbacteria are high effective for their cooperation action. So how to exploit the enrichment is a question worth being study and discussion. In this article, some research was done about the enrichment and try to find new way to study the degrading of toxic compound.The soil polluted by HCH was collected and enriched in the laboratory. Four isomers of HCH(5vppm) could be degraded by enrichment in 36h at 30℃ with and 5% inoculation. No pure culture was got from the enrichment, so some study was done to exploit the enrichment for use in bioremediation. Study shows the enrichment could be amplified as pure culture. The time of formation of enrichment by seeding tank and fermenter are 28h and 20h under conditions of 30℃,300 r/min rotation,with 5% and 10% inoculation, 6L/min and 60L/min aeration.The total DNA from polluted soil and enrichment was extracted and purified to adapt the study of molecular biology. The total DNA was used as template for PCR amplification with conserved lin genes primer. Five genes involved in the degrading of HCH was cloned from total DNA of enrichment, such as linN, linB1, linC1, linD1, linE1, and sequenced. They all shows more than 98% homogenous to the genes got from pure culture in published papers. The G+C content of the linN gene is considerably lower than that of the other genes and the genomic DNA of the S. paucimobilis strains, suggesting that the linN gene may have originated from another organism with a lower G+C content, this may be the result of the gene level transfer. There are at least two pure culture in更多还原