Institutional Repository of Key Laboratory of Marine Ecology & Environmental Sciences, CAS
| 3株南极海洋石油烃低温降解菌（Shewanella sp. NJ49、Pseudoalteromonas sp. NJ289和Planococcus sp. NJ41）基因组学及比较研究 | |
刘芳明
| |
| 学位类型 | 博士 |
| 导师 | 肖天 |
| 2016-04-26 | |
| 学位授予单位 | 中国科学院大学 |
| 学位授予地点 | 北京 |
| 学位专业 | 海洋生态学博士 |
| 关键词 | 南极 烷烃 芳香烃 生物降解 降解机制 适冷 耐盐 抗辐射 全基因组 |
| 摘要 | 本研究对3株南极海洋石油烃低温降解菌Shewanella sp. NJ49、Pseudoalteromonas sp. NJ289和Planococcus sp. NJ41的基因组展开测序、拼接和组装，通过全基因组测序，获得了3株细菌的基因信息，基于目前的基因数据库、蛋白数据库信息，对获得的全基因组进行功能注释，并根据代谢途径注释结果，分析低温降解菌的代谢过程和相关低温、耐盐和抗辐射适应机制和降解酶基因，同时对3株细菌的比较基因组学进行了研究，初步探明了3株细菌的全基因组序列和适应低温、高辐射、高盐极端环境的基因。主要的研究结果如下： （1）Shewanella sp. NJ49基因组总长度约为4.7M，平均G+C含量为41.57%，有4个scaffold；在该基因组中存在5个不完整的前噬菌体；基因组中有12段CRISPR序列，推测该菌在抵抗外源DNA入侵方面具有一定的免疫能力。393蛋白编码基因中存在信号肽序列，占总蛋白编码基因数量的 9.41%；4,173蛋白编码基因中存在至少一个及以上的跨膜螺旋区域，占总蛋白编码基因数量的 25.23%；GO注释结果表明，胞外组分、生物合成过程及细胞组分中的基因最多；COG注释结果表明，一般功能基因居多，其次为未知功能的基因，转录和信号转导过程涉及的基因数量也较多。注释结果表明菌株NJ49存在可降解苯、萘、二甲苯、苯乙烯、氯代烷烃、氨基苯甲酸及芳香族化合物等异源物质的基因。 （2）Pseudoalteromonas sp. 289基因组总长度约为3.5M，平均G+C含量为40.83%；357个蛋白中存在信号肽，占总蛋白数量的9.94%；853个蛋白编码基因中存在至少一个及以上的跨膜螺旋区域，占总蛋白编码基因数量的23.7%；GO注释结果表明，生物过程、胞外氮组分和铁结合蛋白中的基因最多；COG注释结果表明，未知基因最多，高达28.55%，说明该细菌含有大量未知功能的基因，是一个潜在的新基因库，其次为一般功能基因，氨基酸转运和代谢过程涉及的基因数量也较多；KEGG注释结果表明，该菌中代谢类基因最多，以一般代谢类基因最多，其次为氨基酸代谢基因，再次为碳代谢相关基因。系统发育分析结果表明，菌株NJ289可能是一个新种。 （3）Planococcus sp. NJ41基因组总长度3.1M，平均G+C含量为39.69%，该菌共有3,138个编码基因，其总长度2,743,959bp，编码基因的平均长度874bp，编码序列的覆盖度达到87.17%。NJ41基因组内共有127个重复序列，其中LTR长末端重复序列74个，DNA转座子15个，LINE长散在重复序列24个，SINE短散在重复序列10个，RC滚环1个，未知重复序列3个，串联重复序列中小卫星DNA85个，微卫星DNA2个；非编码RNA中，tRNA共67个，平均长度77bp，5s rRNA9个，平均长度115bp，16s rRNA9个，平均长度1,538bp，23s rRNA9个，平均长度2,932bp，sRNA2个，平均长度98bp；NJ41不含有前噬菌体和CRISPRs，但有7个基因岛，平均长度为15,429bp，显示了其部分基因横向起源迹象，该菌的低温适应性或者低温降解功能可能与基因岛上的基因有关；GO注释结果表明，在细胞组分大类基因中细胞和细胞组分的基因数量较多为821个基因，在参与生物过程大类基因中参与参与代谢和细胞的基因数量很多，分别为1,246和1,173个基因，在分子功能大类基因中，催化活性和结合基因数最多，分别为1,319和965个。经过深入分析，在NJ41菌基因组中发现了双加氧酶和单加氧酶相关基因，其中单加氧酶基因有Pcp、次氮基三乙酸酯单加氧酶组分A和组分B、五氯酚-4单加氧酶，双加氧酶基因有联苯双加氧酶、mhqO\色氨酸-2，3-双加氧酶、2-硝基甲烷双加氧酶和PF00903蛋白家族。COG注释表明，参与基本代谢（Metabolism）的蛋白数目最多。二级分类中，COG类别E （氨基酸转运与代谢）、J（翻译，核糖体结构和生物合成）和P（无机离子转运与代谢），所涉及的蛋白数目相对较多，分别占预测蛋白总数的13%、8%、8%。将菌株NJ41基因组预测的所有基因与KEGG数据库进行比对，在7205个全基因中，57%（1790个）的基因与KEGG数据库基因匹配，1790个基因共涉及142条33类KEGG代谢途径。注释结果表明菌株NJ41中存在可以降解苯、萘、二甲苯、苯乙烯、氯代烷烃、氨基苯甲酸及芳香族化合物等异源物质的基因。 （4）3株细菌有各自不同的低温适应、耐盐和抗辐射机制以及降解酶系统。在低温适应机制方面，3株细菌都含有低温适应的分子伴侶GrpE和热激蛋白，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的热激蛋白及热激蛋白转录因子非常丰富且多元化，其分子量有12KDa、70kDa、10kDa不等，还注释到热休克蛋白转录因子hsf1。NJ41菌株中发现冷休克蛋白CspA和CspC和一些类似冷激蛋白的基因和分子伴侣基因（DnaJ、DnaK、DnaE）；抗辐射机制方面，3株细菌均依赖UvrABC系统进行DNA损伤修复，菌株NJ49和菌株NJ289还注释到调节因子UvrY，与Schizosaccharomyces属细菌和Bacillus属细菌中的紫外DNA修复核酸内切酶相近；耐盐机制方面， 3株细菌都包含Trk系统和Na+/H+逆向转运蛋白系统。NJ289还包含prop系统，菌株NJ41含有脯氨酸合成基因：proA、proB及proC和putP、panF等转运体；在降解异源物质方面，3株细菌都可以降解一定范围的芳烃，都含有多种降解酶系统，其中NJ49中注释到烷烃单加氧酶基因alkB和双加氧酶基因，NJ289中注释到了醛脱氢酶、醇脱氢酶等烷烃代谢相关酶基因，NJ41中注释到了儿茶酚-2,3-双加氧酶和乙醛脱氢酶等萘代谢相关酶基因。 （5）3株细菌与其他同属菌存在染色体基因组序列重排现象。系统发育分析预示着NJ289可能是新种，在NJ289中注释到了两个基因簇，显示NJ289在抗菌、抗肿瘤或抗真菌药物合成中具有一定的潜力。在NJ41中注释到有2个基因簇（terpene）。 |
| 其他摘要 | The sequencing, splicing and assembling of genomes from three strains of Antarctic marine Cold-adapted petroleum hydrocarbon-degrading bacteria, including the Shewanella sp. NJ49, the Pseudoalteromonassp. NJ289 and the Planococcus sp. NJ41, were performed in this study. The genetic data of the three strains of bacteria was gained through complete genome sequencing. And based on the existing genetic and protein databases, all the obtained complete genomes were annotated with their functions. Moreover, in reference with the metabolic pathways annotations, the metabolic process of the low temperature degrading bacterium were analyzed, along with its related adaptive mechanisms of low temperature resistance, salt tolerance and radiation proof, as well as its degrading enzyme gene. The main research results were summarized as follows: 1.The total length of Shewanella sp. NJ49 genome was about 4.7M with an average G+C of 41.57% and four scaffolds. In this genome, there existed three complete prophases and one uncertain prophase; signal peptide was identified in 393 protein-coding genes, accounting for 9.41% of its total number; at least one transmembrane helix regions existed in 4,173 protein-coding genes, 25.23% of its total number. GO annotating result suggested most genes existed in the extracellular fraction, biosynthesis process and cellular component and COG annotating result showed that genes of general functions were in the majority, followed by genes of unknown functions and besides genes involved in the processes of transcription and signal transduction were also in large quantity. It was also indicated that such xenobiotic genes as degradable benzene, naphthalene, dimethylbenzene, styrene, chloralkane, aminobenzoic acid and aromatic compounds were found in Shewanella sp. NJ49. 2. The total length of Pseudoalteromonas sp. 289 genome was about 3.5M with an average G+C of 40.83%; neither CRISPRs nor bacteriophage was found in this genome while signal peptide was discovered in 357 proteins, accounting for 9.94% of its total protein number. At least one transmembrane helix region was contained in 853 protein-coding genes, 23.7% of its total number. GO annotating result suggested that most genes existed in the biological process, extracellular nitrogen components and ferritin; COG annotating result showed that the genes of unknown functions were in the majority reaching 28.55%, meaning that there were plenty of unknown functional genes in this bacterium, making it a potential new gene bank. Genes of general functions were in the second place and genes involved in amino acid transport and metabolic process were also in large quantity. KEGG annotating result suggested that metabolism related genes were in the majority with general metabolism related genes at most, followed by amino acid metabolizing genes and next was the carbon metabolizing genes. Phylogenetic analysis indicated that Pseudoalteromonas sp. 289 might be a new kind of bacteria. 3. The total length of Planococcus sp. NJ41 genome was 3.1M with an average G+C of 39.69%. Three completed prophages and one uncertain prophage were included in this genome. There were 3,138 coding genes with an overall length of 2,743,959bp and an average length of 874bp, and its coding sequence coverage reached 87.17%. In NJ41 genome, there were 127 repetitive sequences in all, among which 74 repetitive sequences were arranged in long terminal repeat (LTR). There were 15 DNA transposons, 24 long interspersed elements (LINE) and 10 short interspersed elements (SINE) in the repetitive sequences, one rolling circle (RC), and three unknown repetitive sequences. In tandem repeat sequences, there were 85 minisatellite DNA and two microsatellite DNA. In non-coding RNA, there were sixty-seven tRNA in all with an average length of 77bp; nine 5s rRNA with an average length of 115bp; nine 16s tRNA with an average length of 1538bp; nine 23s rRNA with an average length of 2932bp; two sRNA with an average length of 98bp. Neither prophage nor CRISPRs was found in NJ41, but there were 7 genomic islands with an average length of 15,429bp, showing the transverse origins of partial genes, which suggested that the low temperature adaptability or the low temperature degradable functions might have something to do with genes on the genomic island. GO annotating result told that cell and cellular component genes reached a large number of 821, and there were also a great number of genes engaging in biological process and metabolism, which were 1,246 and 1,173 respectively. Among molecular functional large class genes, catalytic activity genes and combining genes were in the majority, reaching the number of 1,319 and 965 respectively. After in-depth analysis, dioxygenase and monooxygenase related genes were found in NJ41 bacteria genome, among which Pcp, nitrilotriacetatemonooxygenase component A and component B, pentachlorophenol-4 monooxygenase and dioxygenase genes-biphenyl dioxygenase, mhqO\tryphtophan-2, 3- dioxygenase, 2-nitromethane dioxygenase and PF00903 protein family were included in monooxygenase genes. COG annotating result indicated that proteins engaged in general metabolism were at most. In the secondary classification, COG class E (amino acid transport and metabolism), J (transduction, ribosome structure and biosynthesis), and P (inorganic ion transport and metabolism) involved more proteins, accounting for 13%, 8% and 8% of the predicted total protein number respectively. Comparing the predicted genome of bacteria NJ41 with KEGG database, among all the 7,205 whole genes, 57% (1790) genes could be matched with genes in KEGG database, and the 1,790 genes were involved in 142 ways, 33 categories to complete metabolism. Annotating result suggested that xenobiotic genes like degradable benzene, naphthalene, dimethylbenzene, styrene, chloralkane, aminobenzoic acid and aromatic compounds were found in bacterial strain NJ41. 4. The three strains of bacteria had different low temperature resistance, salt tolerance and radiation proof mechanisms and enzyme degrading systems. In respect of low temperature adaptation mechanism, all three strains of bacteria contained low temperature adaptation molecular chaperone GrpE and HSP, and the difference was that there was plenty of cold shock protein in NJ289 and NJ41 while none in NJ49. However, HSP and HSP transcription factors were abundant and polybasic in NJ49, with its molecular weight varying from 12K, to 70K and 10K, and HSP transcription factors hsf1 annotated as well. Cold shock proteins CspA, CspC, and some genes and molecular chaperones genes (DnaJ、DnaK、DnaE) similar to cold shock protein have been found in NJ41 bacteria strains. In terms of radiation proof mechanism, all three strains of bacteria depended on the UvrABC system to achieve wound repair. The regulatory factor UvrY was annotated in bacteria strains NJ49 and Nj289, which was similar to ultra violet DNA repair endonuclease in Schizosaccharomyces bacteria and Bacillus bacteria. In respect of salt tolerance mechanism, all three strains of bacteria included Trk system and Na+/H+ reverse cholesterol transport protein system. The NJ289 also contained prop system while bacterial strain NJ41 included proline biosynthesis genes: transporters i.e., proA，proB, proC and putP. Regarding the degradation of xenobiotics, all three strains of bacteria can degrade certain aromatic hydrocarbons, with their varieties of degrading enzyme systems. 5.The three strains of bacteria proved the existence of chromosomal rearrangement of genomic sequence in comparison with other bacteria of the same genus. Phylogenetic analysis indicated that NJ289 might be a new species. Two gene clusters existed within NJ289, which led to some potential in synthesising antibacterial, antitumor or antifungal drugs. And two gene clusters (terpene) were found in NJ41. |
| 语种 | 中文 |
| 文献类型 | 学位论文 |
| 条目标识符 | http://ir.qdio.ac.cn/handle/337002/112539 |
| 专题 | 海洋生态与环境科学重点实验室 |
| 作者单位 | 中国科学院海洋研究所 |
| 推荐引用方式 GB/T 7714 | 刘芳明. 3株南极海洋石油烃低温降解菌（Shewanella sp. NJ49、Pseudoalteromonas sp. NJ289和Planococcus sp. NJ41）基因组学及比较研究[D]. 北京. 中国科学院大学,2016. |
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