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题名: 水体中甾体激素污染物生态修复技术研究
作者: 桑莹莹
学位类别: 博士
答辩日期: 2011
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 吴钢
关键词: 海洋菌株H5 ; Marine bacterium strain H5 ; 甾体激素的降解 ; Steroid hormones degradation ; 羧酸酯酶 ; Carboxylesterase ; 3-甾酮-△1-脱氢酶 ; 3-Ketosteroid-delta-1-dehydrogenase
其他题名: Study on the Ecological Restoration Technology of Steroid Contaminants in Waterbodies
中文摘要:       水体中存在的甾体激素正潜在威胁着各种动物的种群动态和人类的公共健康。水体中的环境激素已被报道与性发育异常和某些动物的异常雌性化现象相关。因此,人类急需找到一些新技术来降解环境中的甾体激素。德国基尔大学的研究人员在德国基尔市波罗的海一个港湾中分离出了能够降解激素类的菌株H5。 本研究中,通过16S rRNA分析表明海洋菌H5属于γ-变形菌纲,弧菌科(Vibrionaceae),弧菌属(Vibrio)。同时通过3H 示踪标记实验,即,测定培养时间5小时和20小时残留在菌株H5培养基中的甾体激素,可再次验证海洋菌H5能降解包括睾丸酮和雌激素在内的甾体激素。 鉴于3α-羟脱氢酶/羰基还原酶是菌株Comamonas testosteroni代谢甾激素类化合物的关键酶,本研究将其作为阳性对照建立了一个宏基因组系统,以此分离鉴定菌株H5中甾体激素降解的相关基因。本研究在该宏基因组系统中建立了一个菌株H5的染色体DNA片段库, 并把H5染色体DNA片段克隆到了含有增强绿色荧光蛋白报告基因的载体质粒pKEGFP-2中进行荧光检测。同时利用一个含有3α-羟脱氢酶/羰基还原酶基因的报告质粒pK3α-4.6-EGFP3 作为阳性对照。当阳性对照质粒pK3α-4.6-EGFP3,阴性对照质粒pKEGFP-2,克隆有海洋菌H5染色体片段的样品质粒转入宿主细胞大肠杆菌HB101中后,利用甾体激素诱导培养后,荧光酶标仪能检测到甾体激素对蛋白的诱导表达,并筛选出满足条件的阳性质粒。 利用上述pKEGFP-2宏基因组方法,本研究在海洋菌H5中分离发现了两个雌二醇诱导基因。很明显,这两个诱导基因都参与了环境中的雌二醇降解代谢过程。经过测序和在NCBI数据库中的对比分析发现,其中一个基因与分支杆菌属的3-甾酮-△1-脱氢酶基因有关,而另一个基因与细菌Sebadella termitidis 和Brachyspira murdochii 的羧酸酯酶基因具有非常高的相似性。3-甾酮-△1-脱氢酶和羧酸酯酶都是甾体类化合物降解的起始酶。 在本研究中,3-甾酮-△1-脱氢酶基因和羧酸酯酶基因已分别被亚克隆到pET38-12和pET24-17质粒中,在大肠杆菌菌株BL21(DE3)pLysS中超表达带有N-末端His-Tag片断的蛋白。经异丙基-β-D-硫代吡喃半乳糖苷诱导表达后,用镍柱对3-甾酮-△1-脱氢酶和羧酸酯酶进行蛋白纯化,并利用(Bradford方法)进行蛋白定量后,取3-甾酮-△1-脱氢酶蛋白(0.48mg/ml)和羧酸酯酶蛋白(1.28mg/ml)制备抗体,用于进一步研究蛋白与甾体激素降解的关系。 此外,本研究还在菌株H5分离得到了大小为480 bp 的DNA 片段。利用这个DNA片段可以通过PCR技术对未知样品中菌株H5进行鉴定和定量分析。 目前,本研究已经对能够降解甾体激素的海洋菌H5进行了鉴定和系统分类。证明了海洋菌H5可以降解海水中的甾体激素;并在菌株H5染色体DNA中分离出了两雌激素代谢的相关基因,纯化和定量了相应蛋白;基于PCR技术开创了一种新的方法来检测和量化菌株H5。总之,能够降解利用甾体激素的海洋菌 H5将会在水体中甾体激素污染物的修复过程中起到重要的作用。
英文摘要:
      The presence of steroid hormones in the aquatic environment is potentially threatening the population dynamics of many species of animals and public health. Environmental estrogens in water have been reported to be associated with abnormal sexual development and abnormal feminizing responses in some animals. New approaches for the bioremediation of steroid hormones from the environment are therefore urgently sought. The researchers at Kiel University have previously isolated a steroid degrading bacterial strain (H5) from the Baltic Sea, at Kiel, Germany. In the present investigation, 16S rRNA analysis showed that marine strain H5 belongs to the genus Vibrio, family Vibrionaceae and class Gamma-Proteobacteria. Bacterial strain H5 can degrade steroids such as testosterone and estrogens, which was shown in this study by determining the 3H labeled steroid retaining in the bacterial H5 culture medium at incubation times of 5h and 20h. Since 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) is a key enzyme in steroid degradation in Comamonas testosteroni, a meta-genomic system with the 3α-HSD/CR gene as a positive control was established to enable identification of steroid inducible genes from bacterial strain H5. In this meta-genomic system, a library of H5 chromosomal DNA fragments cloned into a fluorescent reporter pKEGFP-2 was constructed. A reporter plasmid pK3α-4.6-EGFP3 containing the 3α-HSD/CR gene was created as a positive control. Steroid induction could be detected by a microplate fluorescence reader, when the plasmids were transformed into Escherichia coli HB101 cells. With this meta-genomic pKEGFP-2 approach, two estradiol-inducible genes were identified from marine strain H5, which are obviously involved in steroid degradation. Sequencing of the pKEGFP-2 inserts and database research at NCBI revealed that one gene corresponds to 3- ketosteroid-delta-1-dehydrogenase from several Mycobacterium strains, while the other showed high similarity to carboxylesterase in Sebadella termitidis and Brachyspira murdochii. Both 3-ketosteroid-delta- 1-dehydrogenase and carboxylesterase are one of the first enzymes in steroid degradation. The 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase genes were subcloned into plasmids pET38-12 and pET24-17, respectively. Overexpression in Escherichia coli strain BL21(DE3)pLysS cells resulted in corresponding proteins with an N-terminal His-tag sequence. After induction with Isopropyl-β-D-Thiogalactopyranoside, 3-ketosteroid-delta-1-dehydrogenase and carboxylesterase were purified in one-step using nickel-chelatechromatography. After protein determination, 3-ketosteroid -delta-1-dehydrogenase (0.48mg/ml) and carboxylesterase (1.28mg/ml) were used to prepare antibodies to determine steroid binding specificity in future research. In addition, a strain H5 specific DNA sequence of 480 bp was identified, which allows sensitive PCR detection, and quantification of strain H5 bacteria in “unknown” seawater samples. Currently, the exact characterization and systematic classification of the marine steroid degrading bacterial strain H5 is envisaged. This study has shown that the marine strain H5 could metabolize steroids; have isolated two estradiol inducible genes from strain H5 chromosomal DNA; purified the corresponding proteins for further research and built a new method to detect and quantify strain H5 by PCR approach. In summary, the strain H5 will play an important role for the bioremediation of steroid contaminations in seawater.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/35086
Appears in Collections:城市与区域生态国家重点实验室_学位论文

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Recommended Citation:
桑莹莹. 水体中甾体激素污染物生态修复技术研究[D]. 北京. 中国科学院研究生院. 2011.
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