【摘要】：隨著對多氯聯(lián)苯(PCBs)生物降解過程中代謝動力學和代謝機理的研究日趨成熟,人們對多氯聯(lián)苯代謝菌的研究逐漸轉移到了基因組和轉錄組水平上,基因組和轉錄組研究不僅能夠從根本上揭示特定生物學過程的分子機理,還能直觀監(jiān)測整個細胞代謝特征,進而獲得聯(lián)苯/PCBs脅迫下,微生物相關細胞生理調節(jié)反應和聯(lián)苯通路可能存在的輔助機制信息。為了研究不同碳源培養(yǎng)條件下,特別是聯(lián)苯條件下紅球菌的細胞轉錄應答,挖掘與多氯聯(lián)苯(PCBs)轉運、代謝及其調控相關的基因,為進一步全面理解PCBs微生物降解的分子機制奠定基礎。本課題以一株多氯聯(lián)苯降解菌紅球菌(Rhodococcussp.R04)為材料,分別提取不同碳源(乙醇、葡萄糖和聯(lián)苯)培養(yǎng)條件下菌體的總RNA,反轉錄合成cDNA。采用高通量測序法分別對這三種樣品進行轉錄組測序,分析測序數(shù)據得出全基因組表達模式,并對不同條件下的基因表達進行差示分析,進而對聯(lián)苯代謝網絡和紅球菌中其他基因的轉錄調節(jié)和代謝應答反應做出相關性分析。并通過Q-RT-PCR分析不同碳源培養(yǎng)條件下的基因表達情況。測序結果表明,與葡萄糖和乙醇相比,聯(lián)苯培養(yǎng)條件下顯著上調(log2 Ratio"g1)基因個數(shù)分別為375和332個。與葡萄糖相比,聯(lián)苯培養(yǎng)條件下相關基因上調表達量與Q-RT-PCR實驗結果基本一致。功能分類獲得細胞組分、分子功能和生物學過程三大類別160多個細小分支的差示表達基因,部分基因參與聯(lián)苯代謝轉錄調控、聯(lián)苯轉運、抗氧化應激反應以及信號傳導通路系統(tǒng)等多種生理過程。參與聯(lián)苯上游代謝途徑的眾多同工酶基因中,只有bphC2和bphDl在聯(lián)苯中大量上調表達,其余同工酶在聯(lián)苯中基本量不變或下調表達。轉錄組注釋及差示分析推測,紅球菌R04中苯甲酸的代謝主要是通過兒茶酚鄰位途徑、間位途徑以及原兒茶酸途徑三條代謝途徑完成。與葡萄糖和乙醇相比,紅球菌R04在聯(lián)苯培養(yǎng)條件下基因表達差異明顯,這為我們進一步解析多氯聯(lián)苯代謝特征和代謝調控提供理論依據。原兒茶酸是多種中藥的主要活性成分和多種藥物的前體物質,其生物學功效具有抗氧化、抗菌、抗腫瘤、抗炎等作用。目前國內市場對原兒茶酸需求量逐年增長,其生產方法主要是采用化學化工法從植物中抽提,提取工藝復雜,在多步提取過程中都有不同程度的損耗,提取率低,且對生態(tài)環(huán)境造成一定破壞。本課題從生物轉化角度解決原兒茶酸生產工藝中的各種問題,首次運用生物酶催化法生產原兒茶酸。將對羥基苯甲酸-3-羥化酶基因重組表達在大腸桿菌BL21(DE3)細胞中,成功構建了一株高效生產原兒茶酸的工程菌,實現(xiàn)了原兒茶酸的綠色催化。此外,本課題對實驗條件進行優(yōu)化以提高原兒茶酸的產率,底物對羥基苯甲酸在每OD菌液中轉化量可達到250～300μg。
[Abstract]:With the study of metabolic kinetics and metabolic mechanism of PCBs (PCBs) biodegradation, the study of PCBs metabolic bacteria gradually transferred to genome and transcriptome level. Genome and transcriptome studies can not only reveal the molecular mechanism of specific biological processes, but also directly monitor the metabolic characteristics of the whole cell, and then obtain biphenyl / PCBs stress. Microorganism related cell physiological regulation response and biphenyl pathway may exist auxiliary mechanism information. In order to study the transcriptional response of erythrococcus cells in different carbon source cultures, especially in biphenyls, the genes related to (PCBs) transport, metabolism and regulation of PCBs were identified. It lays a foundation for further understanding the molecular mechanism of microbial degradation of PCBs. In this study, a polychlorinated biphenyls degrading bacterium erythrococcus (Rhodococcussp.R04) was used as a material to extract total RNA, reverse transcriptional synthesis of cDNA. from different carbon sources (ethanol, glucose and biphenyls). The three samples were sequenced by high throughput sequencing. The whole genome expression pattern was obtained by analyzing the sequencing data, and the gene expression under different conditions was analyzed by differential analysis. The relationship between biphenyl metabolic network and transcription regulation and metabolic response of other genes in erythrococcus was analyzed. Q-RT-PCR was used to analyze the gene expression under different carbon source culture conditions. Compared with glucose and ethanol, the number of up-regulated (log2 Ratio "g1) genes in biphenyl culture was 375 and 332, respectively. Compared with glucose, the up-regulated expression of related genes in biphenyl culture was basically consistent with that of Q-RT-PCR. The differentially expressed genes of more than 160 small branches of cell components, molecular functions and biological processes were obtained by functional classification. Some of the genes were involved in the regulation of biphenyl metabolism and transcription, and the transport of biphenyls. Antioxidant stress response and signal transduction pathway system and other physiological processes. Among the many isozyme genes involved in the upstream metabolic pathway of biphenyls, only bphC2 and bphDl upregulated the expression in biphenyls, while the other isozymes remained unchanged or down-regulated in biphenyls. Transcription notes and differential analysis suggested that the metabolism of benzoic acid in Rhodococcus R04 was mainly accomplished by three metabolic pathways, namely, catechol-adjacent pathway, meso-site pathway and protocatechuic acid pathway. Compared with glucose and ethanol, the gene expression of Rhodococcus rupestris R04 was significantly different from that of glucose and ethanol, which provided a theoretical basis for further analysis of metabolic characteristics and regulation of PCBs. Protocatechuic acid is the main active component of many kinds of traditional Chinese medicine and the precursor of many kinds of drugs. Its biological effects are antioxidant, antibacterial, anti-tumor, anti-inflammatory and so on. At present, the demand for protocatechuic acid in domestic market is increasing year by year. The main production method is to extract from plants by chemical and chemical methods. The extraction process is complex, and the extraction rate is low and the losses are different in the process of multi-step extraction. And caused certain damage to the ecological environment. In this paper, various problems in protocatechuic acid production were solved from the point of view of biotransformation, and protocatechuic acid was first produced by bioenzyme catalysis. The gene of p-hydroxybenzoic acid-3-hydroxylase was expressed in E. coli BL21 (DE3) cells. An engineering strain producing protocatechuic acid was successfully constructed, and the green catalysis of protocatechuic acid was realized. In addition, the experiment conditions were optimized to improve the yield of protocatechuic acid. The conversion of p-hydroxybenzoic acid in the solution of OD could reach 250 渭 g.
【學位授予單位】：山西大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：X172;Q78

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