基于新型邻苯二酚-2,3-双加氧酶基因芘降解假单胞菌的同源重构与时空变化分析

Pyrene is a common polycyclic-aromatic-hydrocarbons-type environmental contaminants. Catechol is an important intermediate product in the degradation pathway of aromatic compound. Catechol 2,3-dioxygenase (B2C23O) catalyzed ring-opening reaction is the rate-limiting step in the degradation of aromatic compounds. In order to significantly improve the degrading capacity of pyrene-degradation bacterium Pseudomonas sp. B2, and acquire a new recombinant strain with strong ecological adaptability, This project is intended to construct the recombinant strain based on the most important gene, B2C23O, in pyrene degradation pathway, and use the origin strain B2 as the host. By a homologous recombination method, the gene units, aromatic-compound-induced Pdmp promoter :: T7 RNA polymerase, and high-efficient promoter PT7 :: B2C23O, were integrated into the genome of the strain B2, to achieve the efficient expression of the B2C23O genes induced by aromatic compounds, and accelerate the metabolism flow of the pyrene degradation pathways. Moreover, based on the novelty and specificity of the B2C23O gene sequence, the variation of the recombinant strain in different years and in different seasons (temporal), in different soil environment and soil layers (spacial) were studied through accurate real-time quantitative PCR (real-time PCR). The conduction of this project could provide new ideas to construct a high efficient aromatic compounds degradation recombinant bacteria, and present scientific support for their environmental release.

芘是常见的多环芳烃类环境污染物。邻苯二酚是芳香族化合物降解的重要中间产物。邻苯二酚2，3-双加氧酶（B2C23O）催化的开环反应是芳香族化合物降解的限速步骤。为了显著提高芘降解菌Pseudomonas sp. B2的降解能力，获得生态适应性强的新型重组菌，本项目拟基于芘降解途径中最关键的B2C23O基因，以本源菌株B2为宿主，通过同源重组的方法，将受芳香族化合物诱导的Pdmp启动子: :T7 RNA聚合酶基因单元和高效启动子PT7:: B2C23O基因单元整合进该菌株的基因组中，实现B2C23O基因受芳香族化合物诱导的高效表达，加快芘降解途径的代谢流；基于该基因的序列的新颖性和特异性，通过精确的实时定量PCR(real-time PCR)对该菌株在不同时间（年份和季节）、不同空间（土壤环境和土层）的变化规律进行研究，为高效芳香族化合物重组菌的构建及环境释放提供新的思路和科学的依据。

芘是常见的多环芳烃类环境污染物。邻苯二酚是芳香族化合物降解的重要中间产物。邻苯二酚2，3-双加氧酶（B2C23O）催化的开环反应是芳香族化合物降解的限速步骤。为了构建能够稳定遗传且高效降解多环芳烃的工程菌，1、通过Over-lap PCR技术扩增出含两种启动子控制的邻苯二酚-2,3-双加氧酶基因，TA克隆连接至PMD-19T载体上，获得两个重组质粒PMD-19T-SPOI-II+C23O和PMD-19T-P2+C23O，转化E.coil Bl21得到两株基因工程菌；2、利用PCR技术对Pseudomonas songnenensis wp3-1的邻苯二酚-2，3-双加氧酶（C23O）基因进行克隆，并将其与自杀性载体pUTmini-Tn5连接,得到重组载体pUTmini-Tn5-C23O。在三亲接合作用下，经mini-Tn5转座将重组载体pUTmini-Tn5-C23O中的C23O基因整合到菌株Pseudomonas sp. wp4的染色体DNA中，最终得到基因工程菌wp4-C23O。3、利用基于液质联用（LC-MS）非靶向的方式研究了三株菌株分别以芘（wp3-1、wp4和I）和山梨醇（S-3、S-4和S-I）为唯一碳源发酵的代谢组学。通过对三株芘降解菌代谢差异物的KEGG通路分析，得到了菌株的总体代谢轮廓图，发现三株菌都存在苯甲酸途径；菌株wp3-1和wp4的天冬氨酸代谢表现为下调，结果分析表明三株菌对芘的主要代谢方式都为水杨酸途径，说明经过基因改造过后的菌株，其代谢途径虽有差异但并未发生很大的改变。4、在克拉玛依油田分别采集深度为5、 20、 50 cm的石油污染土壤样品，测定环境参数；提取石油污染土壤细菌群落基因组DNA，分别构建3个土层细菌16S rRNA基因文库，利用限制性片段长度多态性分析(Restrictionfragment length polymorphisms RFLP)技术初步分群，确定各文库中的代表菌株并测定16S rRNA基因序列；利用软件Biodap计算各群落多样性和丰富度指数，以Neighbor-Joining法构建3个土层细菌的系统发育树；运用软件CANOCO 4.5结合不同样品环境因子的差异进行典型对应分析(CCA)，并探讨了环境因子对细菌多样性的影响。