本文選題：南極 + 烷烴�。� 參考：《中國科學(xué)院研究生院(海洋研究所)》2016年博士論文

【摘要】：本研究對3株南極海洋石油烴低溫降解菌Shewanella sp.NJ49、Pseudoalteromonas sp.NJ289和Planococcus sp.NJ41的基因組展開測序、拼接和組裝,通過全基因組測序,獲得了3株細(xì)菌的基因信息,基于目前的基因數(shù)據(jù)庫、蛋白數(shù)據(jù)庫信息,對獲得的全基因組進(jìn)行功能注釋,并根據(jù)代謝途徑注釋結(jié)果,分析低溫降解菌的代謝過程和相關(guān)低溫、耐鹽和抗輻射適應(yīng)機(jī)制和降解酶基因,同時(shí)對3株細(xì)菌的比較基因組學(xué)進(jìn)行了研究,初步探明了3株細(xì)菌的全基因組序列和適應(yīng)低溫、高輻射、高鹽極端環(huán)境的基因。主要的研究結(jié)果如下:(1)Shewanella sp.NJ49基因組總長度約為4.7M,平均G+C含量為41.57%,有4個scaffold;在該基因組中存在5個不完整的前噬菌體;基因組中有12段CRISPR序列,推測該菌在抵抗外源DNA入侵方面具有一定的免疫能力。393蛋白編碼基因中存在信號肽序列,占總蛋白編碼基因數(shù)量的9.41%;4,173蛋白編碼基因中存在至少一個及以上的跨膜螺旋區(qū)域,占總蛋白編碼基因數(shù)量的25.23%;GO注釋結(jié)果表明,胞外組分、生物合成過程及細(xì)胞組分中的基因最多;COG注釋結(jié)果表明,一般功能基因居多,其次為未知功能的基因,轉(zhuǎn)錄和信號轉(zhuǎn)導(dǎo)過程涉及的基因數(shù)量也較多。注釋結(jié)果表明菌株NJ49存在可降解苯、萘、二甲苯、苯乙烯、氯代烷烴、氨基苯甲酸及芳香族化合物等異源物質(zhì)的基因。(2)Pseudoalteromonas sp.289基因組總長度約為3.5M,平均G+C含量為40.83%;357個蛋白中存在信號肽,占總蛋白數(shù)量的9.94%;853個蛋白編碼基因中存在至少一個及以上的跨膜螺旋區(qū)域,占總蛋白編碼基因數(shù)量的23.7%;GO注釋結(jié)果表明,生物過程、胞外氮組分和鐵結(jié)合蛋白中的基因最多;COG注釋結(jié)果表明,未知基因最多,高達(dá)28.55%,說明該細(xì)菌含有大量未知功能的基因,是一個潛在的新基因庫,其次為一般功能基因,氨基酸轉(zhuǎn)運(yùn)和代謝過程涉及的基因數(shù)量也較多;KEGG注釋結(jié)果表明,該菌中代謝類基因最多,以一般代謝類基因最多,其次為氨基酸代謝基因,再次為碳代謝相關(guān)基因。系統(tǒng)發(fā)育分析結(jié)果表明,菌株NJ289可能是一個新種。(3)Planococcus sp.NJ41基因組總長度3.1M,平均G+C含量為39.69%,該菌共有3,138個編碼基因,其總長度2,743,959bp,編碼基因的平均長度874bp,編碼序列的覆蓋度達(dá)到87.17%。NJ41基因組內(nèi)共有127個重復(fù)序列,其中LTR長末端重復(fù)序列74個,DNA轉(zhuǎn)座子15個,LINE長散在重復(fù)序列24個,SINE短散在重復(fù)序列10個,RC滾環(huán)1個,未知重復(fù)序列3個,串聯(lián)重復(fù)序列中小衛(wèi)星DNA85個,微衛(wèi)星DNA2個;非編碼RNA中,t RNA共67個,平均長度77bp,5s r RNA9個,平均長度115bp,16s r RNA9個,平均長度1,538bp,23s r RNA9個,平均長度2,932bp,s RNA2個,平均長度98bp;NJ41不含有前噬菌體和CRISPRs,但有7個基因島,平均長度為15,429bp,顯示了其部分基因橫向起源跡象,該菌的低溫適應(yīng)性或者低溫降解功能可能與基因島上的基因有關(guān);GO注釋結(jié)果表明,在細(xì)胞組分大類基因中細(xì)胞和細(xì)胞組分的基因數(shù)量較多為821個基因,在參與生物過程大類基因中參與參與代謝和細(xì)胞的基因數(shù)量很多,分別為1,246和1,173個基因,在分子功能大類基因中,催化活性和結(jié)合基因數(shù)最多,分別為1,319和965個。經(jīng)過深入分析,在NJ41菌基因組中發(fā)現(xiàn)了雙加氧酶和單加氧酶相關(guān)基因,其中單加氧酶基因有Pcp、次氮基三乙酸酯單加氧酶組分A和組分B、五氯酚-4單加氧酶,雙加氧酶基因有聯(lián)苯雙加氧酶、mhq O\色氨酸-2,3-雙加氧酶、2-硝基甲烷雙加氧酶和PF00903蛋白家族。COG注釋表明,參與基本代謝(Metabolism)的蛋白數(shù)目最多。二級分類中,COG類別E(氨基酸轉(zhuǎn)運(yùn)與代謝)、J(翻譯,核糖體結(jié)構(gòu)和生物合成)和P(無機(jī)離子轉(zhuǎn)運(yùn)與代謝),所涉及的蛋白數(shù)目相對較多,分別占預(yù)測蛋白總數(shù)的13%、8%、8%。將菌株NJ41基因組預(yù)測的所有基因與KEGG數(shù)據(jù)庫進(jìn)行比對,在7205個全基因中,57%(1790個)的基因與KEGG數(shù)據(jù)庫基因匹配,1790個基因共涉及142條33類KEGG代謝途徑。注釋結(jié)果表明菌株NJ41中存在可以降解苯、萘、二甲苯、苯乙烯、氯代烷烴、氨基苯甲酸及芳香族化合物等異源物質(zhì)的基因。(4)3株細(xì)菌有各自不同的低溫適應(yīng)、耐鹽和抗輻射機(jī)制以及降解酶系統(tǒng)。在低溫適應(yīng)機(jī)制方面,3株細(xì)菌都含有低溫適應(yīng)的分子伴O禛rp E和熱激蛋白,不同的是NJ289和NJ41含有大量的冷激蛋白,NJ49不含有冷激蛋白,但NJ49的熱激蛋白及熱激蛋白轉(zhuǎn)錄因子非常豐富且多元化,其分子量有12KDa、70k Da、10k Da不等,還注釋到熱休克蛋白轉(zhuǎn)錄因子hsf1。NJ41菌株中發(fā)現(xiàn)冷休克蛋白Csp A和Csp C和一些類似冷激蛋白的基因和分子伴侶基因(Dna J、Dna K、Dna E);抗輻射機(jī)制方面,3株細(xì)菌均依賴Uvr ABC系統(tǒng)進(jìn)行DNA損傷修復(fù),菌株NJ49和菌株NJ289還注釋到調(diào)節(jié)因子Uvr Y,與Schizosaccharomyces屬細(xì)菌和Bacillus屬細(xì)菌中的紫外DNA修復(fù)核酸內(nèi)切酶相近;耐鹽機(jī)制方面,3株細(xì)菌都包含Trk系統(tǒng)和Na+/H+逆向轉(zhuǎn)運(yùn)蛋白系統(tǒng)。NJ289還包含prop系統(tǒng),菌株NJ41含有脯氨酸合成基因:pro A、pro B及pro C和put P、pan F等轉(zhuǎn)運(yùn)體;在降解異源物質(zhì)方面,3株細(xì)菌都可以降解一定范圍的芳烴,都含有多種降解酶系統(tǒng),其中NJ49中注釋到烷烴單加氧酶基因alk B和雙加氧酶基因,NJ289中注釋到了醛脫氫酶、醇脫氫酶等烷烴代謝相關(guān)酶基因,NJ41中注釋到了兒茶酚-2,3-雙加氧酶和乙醛脫氫酶等萘代謝相關(guān)酶基因。(5)3株細(xì)菌與其他同屬菌存在染色體基因組序列重排現(xiàn)象。系統(tǒng)發(fā)育分析預(yù)示著NJ289可能是新種,在NJ289中注釋到了兩個基因簇,顯示NJ289在抗菌、抗腫瘤或抗真菌藥物合成中具有一定的潛力。在NJ41中注釋到有2個基因簇(terpene)。
[Abstract]:In this study, the genome of 3 Antarctic marine petroleum hydrocarbon degrading bacteria Shewanella sp.NJ49, Pseudoalteromonas sp.NJ289 and Planococcus sp.NJ41 were sequenced, spliced and assembled. The gene information of 3 strains of bacteria was obtained by whole genome sequencing. Based on the current gene database and protein database information, the whole genome was obtained. According to the annotation of metabolic pathways, the metabolic processes of low temperature degrading bacteria and related low temperature, salt tolerance and radiation resistance mechanism and degrading enzyme genes were analyzed, and the comparative genomics of 3 strains of bacteria was studied. The sequence of the total base group of 3 strains of bacteria and the adaptation to low temperature, high radiation, and salt extreme rings were preliminarily identified. The main research results are as follows: (1) the total length of the Shewanella sp.NJ49 genome is about 4.7M, the average G+C content is 41.57%, and there are 4 scaffold; there are 5 incomplete pre phages in the genome, and there are 12 CRISPR sequences in the genome, which speculates that the bacterium has certain immunity.393 protein in resistance to exogenous DNA invasion. There was a sequence of signal peptide in the encoding gene, accounting for 9.41% of the number of total protein encoding genes, and at least one and more transmembrane helix regions in the 4173 protein encoding gene, accounting for 25.23% of the number of total protein encoding genes. The GO annotation results showed that the extracellular components, biosynthesis process and the gene in the cell components were the most; the COG annotation results showed that The general function gene is many, followed by the unknown function gene, and the number of genes involved in the process of transcription and signal transduction is also more. The annotated results show that strain NJ49 has genes that degrade benzene, naphthalene, xylene, styrene, chloroalkanes, amino benzoic acid and aromatic compounds. (2) Pseudoalteromonas sp.289 genome total The length was about 3.5M, the average G+C content was 40.83%, the 357 proteins had signal peptides, which accounted for 9.94% of the total protein number, and the 853 protein coding genes had at least one and more transmembrane helix regions, accounting for 23.7% of the total protein encoding genes, and the GO annotation results showed that the genes in the biological process, the extracellular nitrogen components and the iron binding proteins were the most important genes. The COG annotation results showed that the unknown genes were the most, up to 28.55%, indicating that the bacteria contained a large number of unknown genes, a potential new gene pool, followed by general functional genes, and the number of genes involved in amino acid transport and metabolism. The KEGG annotation showed that the most metabolic genes in the bacteria were generally metabolized. The gene was the most gene, followed by the amino acid metabolism gene and carbon metabolism related gene again. The phylogenetic analysis showed that the strain NJ289 may be a new species. (3) the total length of Planococcus sp.NJ41 genome was 3.1M, the average G+C content was 39.69%. The total length of the strain was 3138 encoding genes, the total length of the genome was 2743959bp, the average length of the encoding gene was 874bp, The coverage of the coding sequence reached 127 repetitive sequences in the 87.17%.NJ41 genome, including 74 long terminal repeat sequences of LTR, 15 DNA transposons, 24 long scattered sequences, 10 SINE scattered sequences, 1 RC rolling rings, 3 unknown repeat sequences, DNA85, microsatellite DNA2, and non coded RNA in tandem repeat sequences. T RNA is 67, with an average length of 77bp and 5S R RNA9, the average length 115bp, 16S R RNA9, 23S, average length, average length and average length. But there are 7 gene islands, with the average length of the gene, showing the lateral origin of some genes and the low temperature of the bacteria. The function of adaptive or low temperature degradation may be related to genes on the gene island; the GO annotation results show that the number of genes in cell and cell components in the large class of cell components is more than 821 genes, and there are a large number of genes involved in metabolism and cells involved in the major genes involved in biological processes, 1246 and 1173 genes, respectively. In the major molecular function genes, the catalytic activity and the number of binding genes are the most, 1319 and 965 respectively. After in-depth analysis, the dioxygenase and monooxygenase related genes are found in the genome of NJ41 bacteria, in which the monooxygenase genes are Pcp, A and B, pentachlorophenol -4 monooxygenase, dioxygenase, and dioxygenase. Genes with biphenyl dioxygenase, MHQ O tryptophan -2,3- dioxygenase, 2- nitromethane dioxygenase and PF00903 protein family.COG annotation show that the number of proteins involved in basic metabolism (Metabolism) is the most. In the two class classification, COG class E (amino acid transport and metabolism), J (translation, ribosome structure and biosynthesis) and P (inorganic ion transport and substitution) The number of proteins involved is relatively large, accounting for 13% of the total number of predicted proteins, 8%. 8%. compares all the genes predicted by the strain NJ41 genome to the KEGG database. Among the 7205 whole genes, 57% (1790) genes match the KEGG database gene, and the 1790 genes involve 142 33 classes of KEGG metabolic pathways. In strain NJ41, there are genes that can degrade benzene, naphthalene, xylene, styrene, chloroalkanes, amino benzoic acid and aromatic compounds. (4) 3 strains have different low temperature adaptation, salt tolerance and radiation resistance mechanism and degrading enzyme system. In the low temperature adaptation system, 3 bacteria contain the low temperature adapted O RP E NJ289 and NJ41 contain a large number of cold shock proteins, and NJ49 does not contain cold shock proteins, but the heat shock protein and heat shock protein transcription factors of NJ49 are very rich and diverse, and their molecular weights are 12KDa, 70K Da, and 10K Da, and also found that the cold shock protein Csp A and 10K are found in the heat shock protein conversion factor hsf1.NJ41 strain. C and some genes like cold shock proteins and molecular chaperone genes (Dna J, Dna K, Dna E). In the anti radiation mechanism, the 3 strains of bacteria all relied on Uvr ABC system to repair the DNA damage, and the strain NJ49 and strain NJ289 also annotated the regulatory factor. In salt tolerance mechanism, 3 strains of bacteria include the Trk system and the Na+/H+ reverse transporter system.NJ289 also contains the prop system. The strain NJ41 contains the proline synthesis gene: pro A, pro B and pro C and put pro and other transporters; in the degradation of heterologous substances, the 3 bacteria can degrade a certain range of aromatics and contain a variety of degrading enzyme systems. NJ49 was annotated to alkane monooxygenase gene ALK B and double oxygenase gene, and NJ289 was annotated to aldehyde dehydrogenase, alcohol dehydrogenase and other alkanes related enzymes genes. NJ41 was annotated to naphthalene metabolism related enzyme genes, such as catechol -2,3- double oxygenase and aldehyde dehydrogenase. (5) 3 strains of bacteria and other common bacteria have chromosome genome sequence. Column rearrangement. Phylogenetic analysis indicates that NJ289 may be a new species, and two gene clusters are annotated in NJ289, indicating that NJ289 has a certain potential in antibacterial, antitumor, or antifungal drug synthesis. 2 gene clusters (terpene) are annotated in NJ41.

【學(xué)位授予單位】：中國科學(xué)院研究生院(海洋研究所)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：X55;X172

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