3 株南极海洋石油烃低温降解菌（Shewanella sp.NJ49、Pseudoalteromonas sp.NJ28

发布时间：2018-04-21 07:51

本文选题：南极 + 烷烃　；参考：《中国科学院研究生院(海洋研究所)》2016年博士论文

【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,t RNA共67个,平均长度77bp,5s r RNA9个,平均长度115bp,16s r RNA9个,平均长度1,538bp,23s r RNA9个,平均长度2,932bp,s RNA2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、mhq O\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O禛rp E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、70k Da、10k Da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Csp A和Csp C和一些类似冷激蛋白的基因和分子伴侣基因(Dna J、Dna K、Dna E);抗辐射机制方面,3株细菌均依赖Uvr ABC系统进行DNA损伤修复,菌株NJ49和菌株NJ289还注释到调节因子Uvr Y,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:pro A、pro B及pro C和put P、pan F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alk B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)。
[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.

【学位授予单位】：中国科学院研究生院(海洋研究所)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：X55;X172

【参考文献】