【作者】 李子木；

【导师】 闵航；

【作者基本信息】 浙江大学 ， 微生物学， 2008， 硕士

【摘要】 本文以实验室早期分离筛选到的1株二氯喹啉酸高效降解菌Burkholderia cepacia WZ1为供试菌株,运用传统生物学与现代分子生物学技术相结合的方法对菌株WZ1在二氯喹啉酸胁迫下的应激反应机理进行了全面深入的探讨。首先运用传统实验手段观察了菌株在二氯喹啉酸胁迫和非胁迫情况下的生长状况以及检测了该菌株的氧化应激反应,研究了二氯喹啉酸对菌体的影响;再利用双向电泳技术(2-DE)对该菌株在二氯喹啉酸胁迫下全细胞蛋白的表达差异进行分析比较。此外,结合气相质谱技术分析菌株WZ1降解二氯喹啉酸的代谢产物并检测降解关键酶的酶活,从而初步推测二氯喹啉酸的降解途径。最后利用Northern杂交和RT-PCR进一步证实二氯喹啉酸降解过程中的关键酶。本实验阐明了菌株WZ1应对有机污染物胁迫的自我保护体系,初步锁定了二氯喹啉酸生物降解途径中的降解关键酶,为农药污染的生物修复技术提供了理论依据和数据资料。本研究所获得的主要结果如下:1.以二氯喹啉酸高效降解菌洋葱伯克氏菌WZ1(Burkholderia cepacia WZ1)为实验材料,研究了该菌株在二氯喹啉酸胁迫后的生长变化和利用二氯喹啉酸的情况。实验表明,菌株WZ1需经约6h的适应期后才能利用二氯喹啉酸生长。二氯喹啉酸的浓度下降进程与该菌株的生长曲线基本相符合,菌株WZ1在48h后将培养基中的二氯喹啉酸(300μg/L)降解了93.5%。2.基于抗氧化防御系统可随污染胁迫而发生变化,其能间接反映环境中污染物的存在,本文研究了在二氯喹啉酸胁迫下菌株WZ1的氧化应激反应。不同浓度的二氯喹啉酸胁迫下以及同一浓度不同培养阶段的菌株WZ1的超氧化物歧化酶(SOD)表达情况均有差异。经二氯喹啉酸胁迫的菌株可过量表达Fe-SOD酶,并特异性地表达Mn-SOD酶,以形成抗氧化防御系统来保护自我,清除活性氧对细胞的伤害。3.利用双向电泳和蛋白质质谱分析技术研究了二氯喹啉酸胁迫下菌株WZ1的全细胞蛋白表达图谱。通过双向电泳共分离到约500个蛋白质点,分析表明受二氯喹啉酸诱导表达或表达量增强2倍以上的蛋白有69个,另有32种蛋白在二氯喹啉酸胁迫下表达量显著下降甚至消失。对其中的34个蛋白点(采自处理组的26个点以及对照组的8个点)进行了质谱分析,根据这些蛋白质的主要功能可划分为参与氨基酸生成代谢、能量合成、蛋白质合成、解毒和保护功能的蛋白、运输蛋白,参与二氯喹啉酸降解和其他功能蛋白等6大类。核糖体蛋白50S,分子伴侣DnaK以及一些辅助DnaK执行其生理功能的相关蛋白等蛋白在二氯喹啉酸胁迫下表达下调甚至是不表达。这从蛋白表达水平上证明了二氯喹啉酸的确会对菌株WZ1造成伤害。而菌株WZ1则通过分泌热激蛋白70、超氧化物岐化酶、CutA1二价离子耐受蛋白、DNA错配修复蛋白MutS等具保护功能和解毒能力的蛋白质来缓解二氯喹啉酸造成的损害。二氯喹啉酸胁迫下还有多种ABC转运蛋白被诱导表达,这可能与菌株WZ1转运二氯喹啉酸代谢产物或细胞内其他毒害物质的过程有关。4.通过双向电泳实验,还鉴定到了可能参与二氯喹啉酸代谢过程的2个关键酶——邻苯二甲酸双加氧酶(PDR)和氯邻苯二酚1,2-双加氧酶(1,2-CCD)。用传统酶活检测方法测定了这2种酶在含二氯喹啉酸的培养液中菌株不同培养阶段的酶活比,发现这2种酶的酶活变化与菌株WZ1利用二氯喹啉酸的生长情况相符,证明菌株利用二氯喹啉酸的能力与这2种酶的酶活密切相关。通过GC-MS检测到3种可能的二氯喹啉酸中间代谢产物,结合降解过程中的关键酶,初步推测二氯喹啉酸的降解途径为:菌株WZ1首先通过脱羧作用将3,7-二氯喹啉酸转化为3,7-二氯喹啉,经氧化裂解后打开含氮的环,再经过基团转移等作用转化为2-甲酸-5-氯苯胺,转氨作用后形成氯化邻苯二甲酸,然后经过上述测得的邻苯二甲酸双加氧酶和氯邻苯二酚1,2-双加氧酶作用后最终进入三羧酸循环。5.利用Northern杂交和RT-PCR进一步检测二氯喹啉酸的降解关键酶——氯邻苯二酚1,2-双加氧酶。通过设计该酶的引物从菌株WZ1的基因组中扩增到了氯邻苯二酚1,2-双加氧酶的部分基因片段。用该基因片段制备探针后进行Northern杂交,结果证明了该酶的确系由二氯喹啉酸诱导表达。此外,还将该菌株的RNA反转录为cDNA,进行了该酶的cDNA扩增,扩增结果与在菌株WZ1基因组上的扩增结果一致。两个实验从不同的角度在RNA表达水平上证明了菌株WZ1的氯邻苯二酚1,2-双加氧酶由二氯喹啉酸诱导产生,并在二氯喹啉酸的降解过程中发挥着重要作用。更多还原

【Abstract】 An approach combining traditional biological methods and modern molecular biological techniques was used for investigating the stress response of strain Burkholderia cepacia WZ1 upon the quinclorac challenge. The effects of quinclorac were tested by the growth curves and response of superoxide dismutase (SOD) of strain WZ1 under different culture conditions . A proteomic technology-two dimensional electrophoresis (2-DE) was used to separate whole cell proteins of the strain and to identify the differential expressed proteins which were induced by quinclorac. The degrading pathway of quinclorac by strain WZ1 was analyzed based on the results of 2-DE, the GC/MS and enzymes activity assay. What’s more, the enzymes which play important role in the process of quinclorac degradation were confirmed by using the Northern Blot and RT-PCR techniques. The results obtained from this study may give the reference on the application of remedaration technology remedying the pesticide pollution. The main results of this study are as follows:1. Based on the growth of strain WZ1 on different culture media and the various concentrations of quinclorac in the culture medium, it was found that strain WZ1 needed about 6 hours to adapt itself to the stress from quinlcorac, and then it could continue growing slowly. The concentration changes of quinclorac in the culture medium accorded with the growth of the treated strain WZ1, indicating that strain WZ1 indeed could degrade quinclorac for its growing.2. The superoxide dismutase (SOD) of strain WZ1 in response to quinclorac were analyzed because the antioxidant system can be changed according with the stress from the pollutants. The results showed that the SOD activities of strain WZ1 were different when treated with different concentrations of quinclorac and when cells at the different culture phases treated with a certain concentration of quinclorac. The Native PAGE analysis suggested that the strain could express more plentiful Fe-SOD than the normal level and exclusively express Mn-SOD to form a protection wall for itself away from the redox injury.3. An effective and comprehensive method, 2-DE combining with MALDI-TOF/TOF, was used to study the differential proteins induced by quinclorac. About 500 proteins were separated by 2-DE. Exposure to quinclorac altered the global protein expression pattern of B. cepacia WZ1. A total of 69 protein spots were up-regulated (exclusive appearance or at least two-fold expression), while 32 protein spots were down-regulated (disappearance or at least two-fold decrease). 34 protein spots of which were excised from the gels to identify by MALDI-TOF/TOF, 26 spots of which were from treated group and 8 spots from the reference, respectively. Taking into account their main biological functions, these proteins were classified into six different functional groups as amino acid biosynthesis and metabolism, energy synthesis, protein synthesis, detoxification and protection, transport relative, all of proteins may paly roles in the pathway of quinclorac degradation and some other unknowns.Such as 50S ribosomal protein L9, molecular chaperone DnaK and DnaK-related protein were obviously down-regulated or even disappeared, indicating that quinclorac not only influences the normal physiological activities and growth, but also show adverse impact on biosynthesis processes of strain WZ1. The strain protected itself by over-expressing some detoxification and protection proteins, like Mn-SOD, heat shock protein, DNA repair protein, CutAl divalent ion tolerance protein and so on.4. Two differential porteins-phthalate dioxygenase reductase (PDR) and chlorocatechol 1, 2-dioxygenase (1, 2-CCD) were considered as key enzymes in the degrading pathway of quinclorac. The changes of PDR and 1, 2-CCD activities at different culturing time accorded with the growth of B. cepacia WZ1 and the biodegradation rate of quinclorac. It illustrated that the quinclorac degradation and these enzyme activities were closely related. The key enzyme-1, 2-CCD was comfired by Northern Blot and RT-PCR. In addition, there are three products identified by GC-MS, and combining with the key enzymes in the degrading pathway of quinclorac, the degrading pathway of quinclorac may be presumed as follows: quinclorac is transformed into 3, 7-bichlorid quinoline firstly; 3, 7-bichlorid quinoline is catalyzed by dioxygenase, and then shifts to chlorophthalate via aminotransferase reaction. Secondly, chlorophthalate is transformed into 3-chlorocatechol through PDR, phthalate dihydrofiol dehydrogenase and 4, 5-dihydroxyphthalate decarboxylase. Finally, 1, 2-CCD catalyze 3-chlorocatechol into 2-chloro-cis, cis-muconate via an ortho-pathway, the subsequent metabolite is funneled into the TCA cycle.更多还原