【作者】 陈建秋；

【导师】 潘大仁；

【作者基本信息】 福建农林大学 ， 农业生物技术， 2009， 博士

【摘要】 睾丸酮丛毛单胞菌(Comamonas testosteroni)具有降解许多难降解化合物的能力,在污染物的生物修复上具有很高的应用价值。但是,该菌对甾类物质的代谢酶并不是组成型表达的,其表达受到环境中甾类化合物的诱导。因此,该菌对污染物的消化降解速度很慢,限制了该菌在生物修复中的大规模应用。睾丸酮丛毛单胞菌中受睾丸酮诱导表达的调节因子称为teiR基因(testosterone-inducible regulator, teiR),teiR基因在类固醇代谢过程中具有十分重要的意义,可以激活或抑制相关代谢酶基因的表达。因此,本研究通过对teiR基因的克隆及功能分析,从基因转录调控的角度入手,对睾丸酮丛毛单胞菌进行定向调控,提高其降解能力,揭示睾丸酮丛毛单胞菌包括关键酶3a-HSD/CR在内的一系列基因的表达调控机制。该研究对于改造睾丸酮丛毛单胞菌,开发利用睾丸酮丛毛单胞菌进行污染物的环境生物修复等都具有十分重要的意义。研究结果如下:1.根据已报道的睾丸酮丛毛单胞菌调节因子序列设计引物,从睾丸酮丛毛单胞菌ATCC11996菌株中克隆了teiR基因(GenBank登录号:FJ890932)。序列分析表明,其核苷酸序列与已发表的teiR基因(GenBank登录号:AY363220)和tesR基因(GenBank登录号:AB186487)的同源性分别为99 %和95 %。保守结构分析表明,该基因编码蛋白属于LuxR转录调控因子,C末端含有一个HTH DNA结合结构域。2.构建了teiR基因表达载体pET28a-teiR ,在大肠杆菌里进行了诱导表达,并根据重组蛋白的组氨酸特性,利用Qiagen公司的Ni-NTA Spin Column进行TeiR融合蛋白的分离纯化。以分离纯化的TeiR融合蛋白为抗原,制备兔抗TeiR多克隆抗体。该抗体特异性良好,效价可达1:10000。3.以质粒pKtac2和pK18为载体,构建了两个teiR基因重组质粒:pKteiR100和pKtac2-teiR。将重组质粒分别转化或与p6(含3a-HSD/CR基因)共转化大肠杆菌(Escherichia coli) HB101中,酶联免疫吸附实验(ELISA)测定TeiR或3a-HSD/CR的表达量。结果表明含tac强启动子的质粒具有更高的转录活性;在大肠杆菌中,teiR基因能够明显诱导3a-HSD/CR的表达。4.构建teiR基因插入失活突变体的同源重组打靶载体pKmut,并根据同源重组原理电击转化野生型睾丸酮丛毛单胞菌,筛选获得睾丸酮丛毛单胞菌teiR基因插入失活突变体C. mut。应用酶联免疫吸附实验(ELISA)和高效液相色谱法(HPLC)分别分析了突变体蛋白表达及其降解特性。结果发现突变体抑制了teiR基因和关键降解酶3a-HSD/CR的表达,同时突变体丧失了以睾丸酮作为碳源进行生长的能力。5.根据同源重组原理,将pKtac2-teiR电穿孔转化野生型睾丸酮丛毛单胞菌,筛选获得睾丸酮丛毛单胞菌teiR基因高效表达菌AMP8,该菌株已提交中国微生物菌种保藏管理委员会普通微生物中心保存,保存号为CGMCC No. 2365,并申请了一项国家发明专利(专利申请号:2008100724597)。1)利用荧光定量PCR检测了3a-HSD/CR和teiR基因的转录水平。从荧光定量PCR检测的结果来看,睾丸酮均能诱导提高AMP8和野生型菌株3a-HSD/CR和teiR基因的转录水平,但同等条件下AMP8基因转录水平要明显高于野生型菌株。普通RT-PCR结果表明,AMP8在提高teiR基因表达的同时也能显著提高关键酶3a-HSD/CR、4-羟基-2-氧戊酸盐醛缩酶(TIP4)和2-羟基戊-2,4-二烯酸盐水合酶(TIP8)的表达,这些酶都与睾丸酮丛毛单胞菌的类固醇代谢途径有关。2)提取不同培养条件下的AMP8细菌总蛋白,酶联免疫吸附实验(ELISA)测定3a-HSD/CR和TeiR的表达量。结果表明,高效表达菌AMP8 TeiR和3a-HSD/CR的表达量都得到了提高,TeiR表达量最高可达野生型菌株TeiR表达量的5.8倍,3a-HSD/CR表达量最高可达野生型菌株3a-HSD/CR表达量的20倍。进一步说明构建的高效表达菌AMP8在大幅提高TeiR表达量的同时,也能显著地提高关键降解酶3a-HSD/CR的表达量。3)根据双向电泳分析结果,从双向电泳凝胶上挖取15个点,进行MALDI-TOF-MS/MS分析,共有8个蛋白得到鉴定,其中6个点为上调表达,2个点为下调表达。绝大部分鉴定蛋白都与睾丸酮丛毛单胞菌类固醇代谢有关。说明加强teiR表达的高效表达菌AMP8与原始菌相比,能够增加类固醇代谢酶的表达。4)对AMP8进行连续传代培养50代,每隔5代提取细菌总蛋白进行3a-HSD/CR和TeiR蛋白的表达检测。结果表明:在添加抗生素培养时,AMP8 TeiR和3a-HSD/CR的表达均稳定在较高的水平;而不添加抗生素进行培养时,其TeiR和3a-HSD/CR的表达起伏较大,总体表达水平比添加抗生素时低。5)利用HPLC和分光光度法对AMP8的睾丸酮和土壤中甾类物质的降解能力进行分析。结果发现AMP8对睾丸酮和土壤中甾类物质的降解能力均优于野生型菌株。更多还原

【Abstract】 Gram-negative bacterium Comamonas testosteroni has the ability to utilize various steriods as a sole carbon and energe source. Therefore it plays an important role in the bioremediation of these stable compounds in the environment. Insterestingly, the steriod metabolic enzymes were not constitutively expressed, but induced by their respective substrates. This limits its wide use in environmental bioremediation. Testosterone inducible regulator, teiR gene controls a wide variety of activities in various biological processes, some as transcriptional activators, but some as repressors. In the present work, we cloned teiR gene and studyed the function to reveal the relationship between the teiR gene and 3a-HSD/CR. Directionally control the Comamonas testosteroni on gene transcriptional regulation level to empower its degradation ability. The results benefit for applying genetically engineered bacteria of C. testosteroni to assimilate stable hormonally active compounds effectively. The results were as follows:1. teiR gene was cloned from genomic DNA of Comamonas testosteroni. Sequence analysis showed that teiR gene (GenBank accession No. FJ890932) shared highest similarity with teiR (GenBank accession No. AY363220) and tesR gene (GenBank accession No. AB186487), 99 % and 95 %, respectively. teiR gene encoding protein was a putative LuxR-type transcriptional regulator, its domain distribution as N-terminal autoinducer binding domain and C-terminal HTH DNA binding domain.2. Plasmid pET28a-teiR was constructed and performed in E. coli to overexpress TeiR protein. The recombinant protein was purified by its His-tag sequence using Ni-NTA Spin Column supplied by Qiagen. TeiR protein, as assessed by SDS-polyacrylamide gel electrophoresis, was used to prepare antibodies against rabbits. The antibodies reacted very well with the native recombinant protein, and the dilution could be 1:10000.3. Then the teiR gene was sucloned into plasmids pKtac2 and pK18 to yield plasmids pKtac2-teiR and pKteiR100. The recombinant plasmids were transformed into competent Escherichia coli HB101, respectively, or with plasmid p6 (containing 3a-HSD/CR gene) to detect the TeiR and 3a-HSD/CR expression levels by enzyme-linked immunosorbent assay (ELISA). Our results proved that tac promoter was much more efficient than the lacZ promoter and that teiR gene could act as an activator for 3a-HSD/CR expression. 4. Recombinant plasmid pKmut was integrated into Comamonas testosteroni chromosome DNA through homologous recombination to generate teiR gene knock-out mutant. Protein expression levels and its degradation ability were determined by enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography (HPLC), respectively. teiR-disrupted mutant inhibited the 3a-HSD/CR and TeiR expression, using the wild type of Comamonas testosteroni as a control. Finally, the mutant lost the ability to use testosterone as a carbon source. Our results confirmed that the teiR gene mediates the steroid metabolism in Comamonas testosteroni and was necessary for the 3a-HSD/CR expression.5. Recombinant plasmid pKtac2-teiR was integrated into Comamonas testosteroni chromosome DNA through homologous recombination to generate teiR gene overexpression mutant AMP8. AMP8 was already stored at China General Microbiological Culture Collection Center (CGMCC NO.2365), and was made a national patent application (Application No. 2008100724597)1) Real time fluorescence quantitative RT-PCR for detecting the expression of teiR and 3a-HSD/CR had been established, in which the wild type of Comamonas testosteroni treated as a control. The results showed that testosterone could increase this two genes expression in the wild type of Comamonas testosteroni as well as AMP8. AMP8 expressed more than that in the wild type of Comamonas testosteroni under the same conditions. Furthermore, the RT-PCR results indicated that AMP8 constitutively expressed 3a-HSD/CR, but also expressed the enzymes involved in the degradation of steroids, such as 4-hydroxy-2-oxovalerate aldolase and 2-hydroxypenta-2,4-dienoate hydratase.2) AMP8 was cultured in different conditions, and proteins were extracted to detect the TeiR and 3a-HSD/CR expression levels by ELISA using the wild type of Comamonas testosteroni as a control. The results showed that AMP8 could produce several folds of TeiR and 3a-HSD/CR compared to that of the wild type of Comamonas testosteroni, and the best was 5.8-folds of TeiR and 20-folds of 3a-HSD/CR compared to the control. Our results confirmed that AMP8 could widely increase TeiR expression, also increase 3a-HSD/CR expression.3) 8 of 15 differentially expressed proteins were identified by MALDI-TOF-MS and database searching, including 6 up-expressed proteins, 2 down-expressed proteins. The most of the identified proteins were founctioned in the catabolism of steroids.4) AMP8 was cultured for 50 days and total cell lysate was extracted per five days to detect the expression level of 3a-HSD/CR and TeiR using ELISA. The results indicated that TeiR and 3a-HSD/CR were in high and stable expression when inoculated with antibiotic in LB medium, but unstable without antibiotic.5) A HPLC and spectrophotometry assays were established to investigate the dagradation ability between AMP8 and the wild type of Comamonas testosteroni. The results showed that AMP8 had better degradation ability than the wild type of Comamonas testosteroni.更多还原