發(fā)布時間：2018-11-16 11:15

【摘要】：多環(huán)芳烴是一類在環(huán)境中分布十分廣泛的持久性有機污染物,具有潛在的致癌、致畸、致突變作用,對生態(tài)環(huán)境和人類生命健康具有巨大的危害。菲具有多環(huán)芳烴的典型結(jié)構(gòu)——K區(qū)和灣區(qū),是研究低分子量多環(huán)芳烴降解機理的模式化合物。熒蒽是一種高分子量多環(huán)芳烴,其分子結(jié)構(gòu)與一些重要的環(huán)境污染物如二苯并呋喃、咔唑和二苯并二VA英等結(jié)構(gòu)相似,常被作為高分子量多環(huán)芳烴降解研究的模式化合物。本實驗室從西南印度洋深海沉積物樣品中分離獲得一株多環(huán)芳烴降解菌Celeribacter indicus P73~T,能夠高效降解菲和熒蒽等多種多環(huán)芳烴。本文以C.indicus P73~T為研究對象,通過基因組分析、轉(zhuǎn)錄組分析、功能基因研究和代謝物檢測等多種手段解析其降解熒蒽和菲的分子機制和代謝途徑,為認識其環(huán)境作用及開發(fā)相關(guān)生物修復技術(shù)提供支撐。采用Solexa雙末端測序技術(shù)獲得了C.indicus P73~T的全基因組序列。C.indicus P73~T全基因組共含有4,969,388堿基對(bp),其中包括一個環(huán)形染色質(zhì)體(4,529,105 bp)和五個質(zhì)粒(大小范圍7053 155,183 bp),基因組DNA的G+C含量為65.74%。C.indicus P73~T基因組包含4827個蛋白編碼基因、2套r RNA基因(5S r RNA、16S r RNA和23S r RNA各2個)和48個t RNA基因,平均基因長度為909 bp,基因編碼密度為88.3%。在4827個蛋白編碼基因中,有3908個基因(80.96%)歸為22個不同的蛋白質(zhì)直系同源簇。C.indicus P73~T基因組中包含至少138個多環(huán)芳烴降解基因,其中包括6個苯環(huán)羥基化雙加氧酶基因和9個苯環(huán)裂解雙加氧酶基因。經(jīng)基因敲除和功能驗證,P73_0346是負責熒蒽、萘、菲等多環(huán)芳烴第一步雙加氧反應的苯環(huán)羥基化雙加氧酶大亞基,是第一個被發(fā)現(xiàn)的熒蒽-7,8-雙加氧酶,也是甲苯/聯(lián)苯雙加氧酶家族(類群IV)中第一個熒蒽雙加氧酶。通過基因組島和基因水平轉(zhuǎn)移的預測分析,發(fā)現(xiàn)C.indicus P73~T通過基因水平轉(zhuǎn)移等方式獲取了一些多環(huán)芳烴降解基因,增強了多環(huán)芳烴降解能力。對C.indicus P73~T轉(zhuǎn)錄組進行了測序分析,發(fā)現(xiàn)1184個基因(24.5%)在熒蒽誘導培養(yǎng)時發(fā)生差異表達,其中975個基因(20.2%)上調(diào)表達。通過基因組和轉(zhuǎn)錄組聯(lián)合分析,發(fā)現(xiàn)至少有110個基因可能參與了熒蒽的降解過程,其中包括趨化、信號感應和傳導、調(diào)控、轉(zhuǎn)運和降解等過程的相關(guān)基因。采用氣相色譜-質(zhì)譜聯(lián)用技術(shù)檢測到C.indicus P73~T降解熒蒽的4種重要中間代謝產(chǎn)物:1-二氫-苊酮、二氫苊醌、1,2-二羥基苊烯和1,8-萘二甲酸。據(jù)此推測C.indicus P73~T通過C-7,8雙加氧途徑降解熒蒽。同時綜合基因組分析結(jié)果、轉(zhuǎn)錄組分析結(jié)果、功能基因研究結(jié)果和文獻報道,推測了C.indicus P73~T降解熒蒽的代謝途徑。C.indicus P73~T降解熒蒽的代謝途徑是經(jīng)由7,8-二羥基熒蒽間位裂解反應的C-7,8雙加氧途徑,中間代謝產(chǎn)物鄰苯二甲酸和原兒茶酸通過β-酮己二酸途徑最終進入三羧酸循環(huán)徹底降解。同時,熒蒽C-7,8雙加氧途徑是C.indicus P73~T降解熒蒽的唯一代謝途徑,因此,C.indicus P73~T可以作為一株研究熒蒽C-7,8雙加氧途徑的模式菌株。采用氣相色譜-質(zhì)譜聯(lián)用技術(shù)檢測到C.indicus P73~T降解菲的2種重要中間代謝產(chǎn)物:1-萘酚和1-羥基-2-萘甲酸,并據(jù)此推測C.indicus P73~T通過C-3,4雙加氧途徑降解菲。綜合代謝組分析結(jié)果、基因組分析結(jié)果、基因功能研究結(jié)果以及已報道的菲代謝途徑,推測了C.indicus P73~T降解菲的C-3,4雙加氧途徑:首先在菲C-3,4位發(fā)生雙加氧反應,隨后逐步氧化形成1-羥基-2-萘甲酸,并進一步脫羧生成1-萘酚。1-萘酚等毒性代謝物在C.indicus P73~T菌體和培養(yǎng)基中大量積累,導致菌體生長微弱。C.indicus P73~T是Celeribacter屬的一個新種,能夠廣譜、高效地降解多種多環(huán)芳烴,在石油泄漏、多環(huán)芳烴污染等海洋受污染環(huán)境的生物修復中具有較大的應用前景。本文研究解析了C.indicus P73~T降解熒蒽和菲的分子機制和代謝途徑,能夠為環(huán)境中熒蒽和菲污染的生物修復技術(shù)提供理論指導。
[Abstract]:Polycyclic aromatic hydrocarbon is a kind of persistent organic pollutant which is widely distributed in the environment, has potential carcinogenic, teratogenic and mutagenic effects, and has great harm to the ecological environment and human health. Phenanthrene has a typical structure of polycyclic aromatic hydrocarbons, the K region and the Gulf region, and is a mode compound for studying the degradation mechanism of low molecular weight polycyclic aromatic hydrocarbons. It is a kind of high molecular weight polycyclic aromatic hydrocarbon, and its molecular structure is similar to some important environmental pollutants, such as dibenzol and dibenzo-dibenzo-dibenzo-2-VA, and is often used as a mode compound for the degradation of high-molecular-weight polycycloaromatics. The laboratory isolated from the deep-sea sediment sample of the southwest Indian Ocean to obtain a multi-cyclic aromatic hydrocarbon degrading bacterium, Celeribacter indicius P73-T, and can effectively degrade various polycyclic aromatic hydrocarbons such as the phenanthrene and the polyaromatic hydrocarbon. In this paper, C. indicus P73-T is used as the research object, and the molecular mechanism and the metabolic pathway of the degradation of the phenanthrene and phenanthrene are analyzed by a variety of means such as genome analysis, transcription group analysis, functional gene research and metabolite detection, so as to provide support for the recognition of its environmental effect and the development of related bioremediation technology. The whole genome sequence of C. indicius P73-T was obtained by using the Solexa double-terminal sequencing technology. .C.indicus P73~T鍏ㄥ熀鍥犵粍鍏卞惈鏈，

本文編號：2335362