【摘要】：作為新興綠色水處理劑,微生物絮凝劑研發(fā)及應(yīng)用越來(lái)越受到各國(guó)科學(xué)家的重視,多年來(lái)一直是環(huán)境生物技術(shù)領(lǐng)域的研究熱點(diǎn)。然而,微生物絮凝劑的生物合成途徑、代謝途徑及相關(guān)基因尚不清晰,致使無(wú)法確定野生菌株產(chǎn)絮性狀不穩(wěn)定和產(chǎn)量低的根本原因,無(wú)法從根本上解決微生物絮凝劑存在的問(wèn)題�；诨蚪M/轉(zhuǎn)錄組分析,本文開(kāi)展了多糖型微生物絮凝劑合成途徑及其群體調(diào)控機(jī)制的研究,為填補(bǔ)微生物絮凝劑合成途徑研究的不足,為解決微生物絮凝劑產(chǎn)量低和產(chǎn)絮性狀不穩(wěn)定問(wèn)題提供理論支持�；贏grobacterium tumefaciens F2的全基因組測(cè)序數(shù)據(jù),采用生物信息學(xué)和比較基因組方法,分析菌株F2基因組中主要代謝途徑與調(diào)節(jié)機(jī)制相關(guān)的功能基因;產(chǎn)絮菌F2所產(chǎn)微生物絮凝劑主要有效成分為多糖,因此將多糖型微生物絮凝劑合成基因集中在多糖合成基因上。在菌株F2基因組中發(fā)現(xiàn)了84個(gè)多糖型微生物絮凝劑合成基因,其中參與琥珀酰聚糖合成exo基因最可能指導(dǎo)多糖型微生物絮凝劑合成;此外,菌株F2質(zhì)粒上發(fā)現(xiàn)了與Rhizobium和Agrobacterium中tra R/tra I群體感應(yīng)系統(tǒng)具有高度相似的基因簇;完成了多糖型微生物絮凝劑合成基因及其調(diào)控基因的解析,為多糖型微生物絮凝劑合成基因和群體感應(yīng)系統(tǒng)調(diào)控基因的功能解析提供大量基因組信息。為了確定多糖型微生物絮凝劑合成基因的功能和合成途徑,對(duì)產(chǎn)絮菌F2發(fā)酵過(guò)程中高通量基因轉(zhuǎn)錄組和基因表達(dá)差異進(jìn)行分析;從基因組解析的84個(gè)多糖型微生物絮凝劑合成基因中確定了65個(gè)在發(fā)酵過(guò)程中發(fā)生顯著差異的基因,通過(guò)比對(duì)A.tumefaciens C58和F2合成胞外多糖的單糖組分和合成基因的異同,構(gòu)建多糖型微生物絮凝劑exo基因簇,并確定了多糖型微生物絮凝劑合成基因的功能和合成途徑,揭示引起多糖型微生物絮凝劑產(chǎn)量和效能差異的合成基因類型,為多糖型微生物絮凝劑合成途徑的研究提供嶄新的思路和方法,為強(qiáng)效表達(dá)合成途徑的關(guān)鍵基因從而實(shí)現(xiàn)多糖型微生物絮凝劑定向合成方法提供理論依據(jù)。細(xì)菌通過(guò)群體感應(yīng)現(xiàn)象協(xié)調(diào)群體基因同步表達(dá)從而調(diào)控胞外物合成的群體行為,而提高調(diào)控基因的表達(dá)可以實(shí)現(xiàn)定向調(diào)控多糖型微生物絮凝劑的合成這種群體行為;基因組/轉(zhuǎn)錄組分析發(fā)現(xiàn)產(chǎn)絮菌F2中具有群體感應(yīng)系統(tǒng)調(diào)控基因,利用UPLC-MS/MS在菌株F2發(fā)酵液中檢測(cè)出七種N-acyl-homoserine lactones(AHLs);同時(shí),研究投加C6-HSL和3-oxo-C8-HSL下對(duì)多糖型微生物絮凝劑合成的影響,研究結(jié)果表明,投加一定濃度的C6-HSL和3-oxo-C8-HSL時(shí),微生物絮凝劑的糖型微生物絮凝劑效能得到了顯著提高,在投加C6-HSL和3-oxo-C8-HSL最優(yōu)投加量0.4μM和0.2μM下,多糖濃度分別提高1.6倍和1.4倍。此外,對(duì)投加C6-HSL和3-oxo-C8-HSL下溫度和初始p H值對(duì)多糖型微生物絮凝劑合成的影響進(jìn)行研究,并通過(guò)響應(yīng)面優(yōu)化得到了C6-HSL和3-oxo-C8HSL投加下微生物絮凝劑發(fā)酵最優(yōu)條件,在此條件下合成的多糖型微生物絮凝劑與未投加相比,多糖濃度分別提高1.75倍和1.55倍,絮凝率分別提高了10%和10.96%。為了進(jìn)一步探究信號(hào)分子AHLs對(duì)多糖型微生物絮凝劑合成途徑的調(diào)控,基于基因轉(zhuǎn)錄組測(cè)序分析,對(duì)C6-HSL投加下菌株F2群體感應(yīng)系統(tǒng)和多糖型微生物絮凝劑合成基因表達(dá)差異進(jìn)行分析;從基因?qū)用孀C實(shí)了菌株F2基因組中存在與Rhizobium高度相似的traR/tra I群體感應(yīng)系統(tǒng),而C6-HSL也可以作為自誘導(dǎo)物與Agau_P200438形成復(fù)合物,參與產(chǎn)絮菌F2多糖型微生物絮凝劑合成的調(diào)控作用,誘導(dǎo)參與多糖轉(zhuǎn)移和轉(zhuǎn)運(yùn)基因的表達(dá)。綜合以上分析,從宏觀和微觀層面揭示了信號(hào)分子AHLs對(duì)多糖型微生物絮凝劑合成的群體調(diào)控機(jī)制,為理解微生物絮凝劑合成的調(diào)控機(jī)制,為提高調(diào)控基因的表達(dá)從而實(shí)現(xiàn)多糖型微生物絮凝劑定向調(diào)控的方法提供理論依據(jù)。
[Abstract]:As an emerging green water treatment agent, the research and application of microbial flocculant is becoming more and more important to national scientists, and has been a hot topic in the field of environmental biotechnology for many years. However, the biosynthetic pathway, metabolic pathway and related gene of microbial flocculant are not clear, so that the root cause of the instability and low yield of the wild strain can not be determined, and the problem of the existence of the microbial flocculant can not be fundamentally solved. Based on the analysis of the genome/ transcription group, this paper has carried out the research of the synthetic route of the polysaccharide-type microbial flocculant and its population control mechanism, and provides the theoretical support for solving the problem of the low yield of the microbial flocculant and the instability of the cotton-producing character. Based on the whole genome sequencing data of the Agrobacterium tumefacens F2, the functional genes related to the main metabolic pathway and the regulation mechanism in the strain F2 genome are analyzed by using the bioinformatics and the comparative genomic method, and the main active components of the microbial flocculant produced by the cotton-producing bacteria F2 are polysaccharides, Thus, the polysaccharide-type microbial flocculant synthesis gene is concentrated on the polysaccharide synthesis gene. in that genome of the strain F2, 84 polysaccharide-type microbial flocculant synthesis genes are found, wherein the synthesis of the exo gene involved in the synthesis of the amber-type microbial flocculant is most likely to guide the synthesis of the polysaccharide-type microbial flocculant; in addition, The strain F2 plasmid was found to have a highly similar gene cluster with the tra R/ tra I group induction system in the Rhizobium and the Agrobacterium, and the synthesis gene of the polysaccharide-type microbial flocculant and the analysis of the regulatory gene thereof were completed. and provides a large number of genome information for functional analysis of a polysaccharide-type microbial flocculant synthetic gene and a population induction system regulation gene. in ord to determine that function and the synthetic route of the synthetic gene of the polysaccharide-type microbial flocculant, the difference between the high-throughput gene transcription group and the gene expression in the fermentation process of the cotton-producing strain F2 is analyzed; 65 of the 84 polysaccharide-type microbial flocculant synthetic genes which were analyzed from the genome were identified and 65 genes which had significant difference in the fermentation process were identified, and the polysaccharide-type microbial flocculant exo gene cluster was constructed by the similarity and difference of the monosaccharide components and the synthetic genes of the extracellular polysaccharide synthesized with A. tumefacens C58 and F2. The function and the synthetic route of the synthetic gene of the polysaccharide-type microbial flocculant are determined, the synthetic gene type which causes the difference of the yield and the efficiency of the polysaccharide-type microbial flocculant is disclosed, and a brand-new method and a method are provided for the research of the synthetic route of the polysaccharide-type microbial flocculant, and provides a theoretical basis for realizing the directional synthesis method of the polysaccharide-type microbial flocculant for a key gene of a strong expression and synthesis route. the bacteria are synchronously expressed by the group induction phenomenon to regulate the group behavior of the extracellular substance synthesis, and the expression of the regulation gene can be improved to realize the synthesis of the directional control polysaccharide-type microbial flocculant; In the genome/ transcriptome analysis, a group induction system control gene was found in F2, and seven N-acyl-homoserine lactuones (AHLs) were detected in the strain F2 fermentation broth by UPLC-MS/ MS, and the effects of the addition of C6-HSL and 3-oxo-C8-HSL on the synthesis of polysaccharide-type microbial flocculant were studied. The results showed that When the concentration of C6-HSL and 3-oxo-C8-HSL was added, the efficiency of the microbial flocculant of the microbial flocculant was significantly improved, and the polysaccharide concentration was increased by 1. 6-fold and 1. 4-fold, respectively, with the optimal dosing of C6-HSL and 3-oxo-C8-HSL. In addition, the effects of the temperature and initial p H on the synthesis of the polysaccharide-type microbial flocculant were studied under the conditions of the temperature and the initial p-H value of the addition of C6-HSL and 3-oxo-C8-HSL, and the optimal conditions for the fermentation of the microbial flocculant under the addition of C6-HSL and 3-oxo-C8HSL were obtained through the response surface optimization. The polysaccharide-type microbial flocculant, which was synthesized under this condition, was increased by 1. 75-fold and 1.55-fold, respectively, and the flocculation rate was increased by 10% and 10.96%, respectively, compared with that of the undosed. in ord to further explore that regulation of the synthetic pathway of the polysaccharide-type microbial flocculant by the signal molecule AHLs, the difference of the expression of the synthetic gene of the strain F2 population induction system and the polysaccharide-type microbial flocculant is analyzed based on the gene transcription group sequencing analysis; A TRAR/ tra I population induction system similar to that of Rhizobium in the genome of the strain F2 was confirmed from the genetic level, and the C6-HSL could also be used as the self-inducer and the Adau _ P200438 to form a complex to participate in the regulation and control of the synthesis of the F2 polysaccharide-type microbial flocculant. The expression of the genes involved in the transfer and transport of the polysaccharides was induced. In the light of the above analysis, the group regulation mechanism of the synthesis of the polysaccharide-type microbial flocculant from the signal molecule AHLs is disclosed from the macro and micro level, and the regulation mechanism for the synthesis of the microbial flocculant is provided. and provides a theoretical basis for improving the expression of the regulation gene so as to realize the directional control of the polysaccharide-type microbial flocculant.
【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：X703.5;TU991.2

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