戈登氏菌R9株高效降解17 beta-雌二醇的作用机制

Estrogen pollution caused by animal industry exhibited strong ecologic toxic effect, which severely influenced the health and development of human, animals and other livings. Until now microorganism biodegradation is one of the best tools to repair the estrogen-polluted areas, thus it is critical to obtain highly effective estrogen-degrading bacteria and elucidate the mechanism of estrogen biodegradation by bacteria. Previously a highly effective estrogen-degrading bacteria Gordonia sp. R9 was isolated and its complete genome sequence was obtained in our lab, and 25 genes which involved in the degradation of estragens were observed. The present study will study the inducible mechanism of 17β-estradiol 17 dehydrogenase (17β-EDH) catabolizing estradiol to estrone in Gordonia sp. R9. Procaryotic expression and purification of protein 17β-EDH and transcriptional repressor EdhR, ELISA, Southern blot, Western blot, homologous recombination, DNase I footprinting and electrophoretic mobility shift assay (EMSA) will be used to confirm the molecular mechanism of effective degradation of E2β in Gordonia sp. R9. The research findings in this study will provide new insights into repairing estrogen-polluted areas caused by animal industry in future.

现代畜禽养殖排放大量雌激素进入环境中，由此产生的生态毒性效应严重影响人、动物及其它生物的健康生长。目前，微生物修复是雌激素污染治理的重要手段之一。因此，获得高效的雌激素降解菌株并解析其降解机制至关重要。课题组前期研究获得了雌激素高效降解菌株—戈登氏菌R9株并对其全基因组序列进行了测定，共发现25个雌激素降解相关基因。本项目拟对戈登氏菌R9株体内降解17beta-雌二醇（E2β）为雌酮（E1）的17β-雌二醇脱氢酶（17β-EDH）被E2β诱导表达的分子机制降解进行研究，利用蛋白原核表达与纯化、ELISA、Southern blot、Western blot、同源重组、DNase I足迹分析法和凝胶漂移实验等技术和方法，拟解析戈登氏菌R9株高效降解E2β的作用机制，为我国开展畜禽养殖环境中雌激素污染的处理提供技术支撑。

雌激素是现代养殖业产生的最重要的持久性污染物之一。微生物修复是其污染治理的重要手段。项目组从动物排泄物污染的水土环境样品中分离纯化到了30余株雌激素高效降解菌，主要包括假单胞菌属、睾丸酮丛毛假单胞菌属、戈登氏菌属、不动杆菌、红球菌等不同种属的微生物。经高效液相色谱鉴定，戈登氏菌R9株是其中降解能力最强的一株，5 mg/L的17beta-雌二醇能在24小时之内被该菌完全降解。该菌对环境适应范围广，在4-40℃以及pH1.0-11.0之间对雌激素均有降解能力。该菌底物利用强，50 μg/L-5 mg/L的雌激素都能被该菌降解，该菌还能够利用E2之外的其它多种固醇激素作为唯一的碳源和能源，且采用GC-MS从该菌体内鉴定出了2个新的代谢中间产物，从而推测出了一条新的E2降解途径。为了深入研究该菌高效降解雌激素机理，利用二代测序技术（illumina）结合三代测序技术（Pacbio）的方式对其进行全基因组测序分析，结果表明该菌基因组成环，全长为6,194,233 bp，共有5,755个基因，GC 含量为67.90%。通过对该菌体内固醇代谢基因分析，发现该菌除了有完整的固醇降解途径外，还有部分固醇激素合成途径中的相关基因，表明该菌体内固醇激素降解途径丰富多样。通过戈登氏菌R9株基因组文库构建，从该菌体内筛选到了6个含有雌激素降解基因的片段以及20多个参与雌激素降解的基因。E2诱导后戈登氏菌R9株转录组学分析，筛选到了72个E2诱导表达基因。通过原核表达与纯化17beta-EDH蛋白，并制备多克隆抗体，Western blot结果表明，E2能够诱导戈登氏菌R9株体内17beta-EDH蛋白上调表达。上述研究成果有助于进一步揭示戈登氏菌R9株高效降解雌激素的分子机制。