【作者】 王晓佳；

【导师】 程安春； 汪铭书；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2013， 博士

【摘要】 鸭传染性浆膜炎是鸭,火鸡和鸟类之间的一种传染性疾病,其病原是鸭疫里默氏杆菌。本病的病死率是90%左右,1-8周鸭是主要的感染鸭,呈急性或慢性败血症,病变表现上以纤维素性心包炎、肝周炎、气囊炎、脑膜炎及部分病例出现关节炎为特征。1993年正式将其确立为一个独立的分支并改名为鸭疫里默氏杆菌。鸭疫里默氏杆菌的血清型共有25种,各个血清型之间没有交叉保护。目前鸭疫里默氏杆菌的基因组遗传信息在NCBI的数据库中仅有5个完整的全基因组序列以及一个不完整的序列片段。其中三株是由本实验室测序完成,分别为标准菌株ATCC11845.血清型一型的RA-CH-1和血清型二型的RA-CH-2.由于基因组序列的数据稀少,有关鸭疫里默氏杆菌的基因组结构情况研究更加稀少,因此我们希望能够通过我们的研究,为鸭疫里默氏杆菌的研究提供一定的基础。我们分离了两株临床株,在ATCC菌种保存库中购买了ATCC11845,然后对三株鸭疫里默氏杆菌进行测序和注释。这三株鸭疫里默氏杆菌和与NCBI数据库中另外一株RA-GD的数据进行比对分析。分析结果显示ATCC11845.RA-GD和RA-CH-2三株序列的基因数目相差不大,GC含量都在35%左右。只有RA-CH-1表现出了比较大的差异,基因组序列长度比另外三株多出了140,000bp,GC含量也略高于其他几株鸭疫里默氏杆菌,基因数目增加了100个以上,与相同血清型的RA-GD相比其差异反而比不同血清型的RA-CH-2更大。基因组所含蛋白种类分析结果显示,鸭疫里默氏杆菌中的功能性蛋白主要有以下几种：1、铁转运蛋白TonB蛋白家族：该家族的主要功能是细菌的铁转运系统,由于铁的吸收是细菌生长至关重要的部分,而且铁对细菌的毒力因子也具有很大的影响力,因此铁运输途径对维持细菌存活非常重要。2、荚膜多糖：荚膜多糖是许多细菌的重要致病因素,该类蛋白不仅能够抑制许多细菌生物膜的形成,同时在一些菌株中也具有破坏已经生成的生物膜的特性,并且在某些细菌中还有加强部分抗生素破坏细胞外膜的效果。3、外排系统蛋白：一个是微小多重耐药(SMR)家族,另一类是主异化超家族(MFS)的MFS-1家族相关的蛋白。4、抗生素抗性相关基因：例如vanz家族蛋白,为万古霉素的抗性基因；青霉素酶阻遏蛋白；吖啶黄耐药蛋白：樟脑耐药蛋白；多重抗生素耐药性相关蛋白。重复序列是基因组中重复出现的核苷酸序列。这些序列一般不编码多肽,重复序列可分为散在重复序列和串联重复序列。散在重复序列又称转座子元件,包括3种：长末端重复序列、短散在重复序列、长散在重复序列。长末端重复序列(LTR)具有调节病毒基因转录、复制以及病毒基因整合等功能。短散在重复序列(SINE)和长散在重复序列(LINE),广泛存在于真核生物中并占据着基因组相当大一部分,它们通过反转座的形式不断插入到基因组染色体的新座位,由于其反转座插入的不可逆性和独立性,它们被认为是研究物种类群系统发育关系和群体遗传学的绝好标记。重复序列在微生物的基因组中有重要作用,因此我们通过对4株菌的重复序列进行了分析。我们发现4株菌的串联重复序列,无论在拷贝数上还是重复序列单元间的一致度上,都保持高度一致。通过对重复序列区域和基因交叠的地区的COG功能富集分析结果表明,菌株ATCC-11845、RA-CH-2、RA-GD的重复序列基因在折叠,重组和修饰方面的功能上显著性富集,而在其他功能上没有出现显著性富集(显著性水平=0.05)；菌株RA-CH-1在所有COG功能上,均为表现出显著性富集。噬菌体(bacteriophage)是感染细菌、真菌、放线菌及螺旋体等微生物的病毒。而整合于宿主菌基因组中的噬菌体则称为前噬菌体(prophage)。根据前人的研究成果,前噬菌体在细菌的生长繁殖和进化过程中均发挥着重要的作用,而且细菌染色体中整合了前噬菌体基因组的现象是非常普遍的。前噬菌体的整合能够提高细菌获取外来有用序列的几率,增强病原菌致病性,或者提高其对生存环境的适应能力。但是,前噬菌体基因的插入也可能导致细菌被裂解死亡。因此,基因组中整合的前噬菌体被认为是研发新型的抗菌药物的重要目标。通过我们的检测,四株鸭疫里默氏杆菌的基因组中都未能发现完整的前噬菌体的存在,仅仅有一些与噬菌体或者前噬菌体相似的序列碎片。基因岛是横向原点证据特性的基因组中的一部分,根据其携带的基因的种类,还可以分为毒力岛、抗性岛、代谢性岛、共生性岛、适应性岛等类型。基因岛拥有共生和致病机制或生物适应性的成簇分布的基因集合片段,其具有特定的结构特征。一般情况下基因岛的GC含量、核苷酸的偏向性以及密码子的使用情况等,相对于其染色体的水平会表现出一定的差异。许多基因岛还会包含部分外源基因,例如噬菌体和质粒的移动元件,可以实现自我剪接后平行移动到其他合适的细菌机体中,这项能力是部分毒力基因或者耐药基因在不同细菌之间传递的重要方法之一,也是造成细菌多重耐药的可能原因之一。我们的结果显示,RA-CH-1拥有10个基因岛,但是都未能在其他三株中找到相似的基因岛,其中的RGL4和RGL9这两个基因岛与鼻气管鸟杆菌DSM15997的序列有相当高的相似性。ATCC11845、RA-GD和RA-CH-2这三株的基因岛彼此之间的相似性较高。每个菌株都有其独特的基因岛。基因家族是指具有共同祖先基因,经过倍增和突变后,形成的一类基因的集合。从基因家族以及同源基因的分析结果来看,RA-CH-1拥有独特的基因家族最多,高达16个,而其他三株只有1到2个。4株鸭疫里默氏杆菌共同拥有的基因家族为630个,其比例大概在菌株所拥有的基因家族数目的二分之一到三分之一之间,这说明鸭疫里默氏杆菌拥有的基因家族种类没有很高的共性,其基因家族的复杂性可能对其的生物学特性造成一定的差异。我们分别对四株鸭疫里默氏杆菌构建了系统发育进化树,保守元件和非保守元件的进化树,通过分析发现他们的保守元件和非保守元件两者的拓扑结构是一致的,只是分支长度不一样,系统发育进化树的分析结果显示菌株RA-CH-1与RA-CH-2最近缘,这两株与ATCC-11845有更亲缘的关系,它们属于同一个分支,而与RA-GD分属于不同的分支。通过对四株鸭疫里默氏杆菌的非保守区域和保守区域都做了分析,结果发现鸭疫里默氏杆菌的非保守区域具有更高的突变速率,其突变速率大约是保守区域的8倍。更多还原

【Abstract】 Duck infectious serositis is a contagious disease of ducks, turkeys and birds, the pathogen is the Riemerella Anatipestifer. The mortality rate of this disease is about90%,1-8weeks ducks are the main infected ducks, the general course of acute or chronic sepsis. Lesions mainly for the cellulose pericarditis, perihepatitis, the air sac inflammation, meningitis, and in some cases also appear arthritis. Officially established as a separate branch in1993, and renamed Riemerella Anatipestifer. Riemerella Anatipestifer have25serotypes, but on cross protection between serotypes. The genomic information Riemerella Anatipestifer only five complete genome sequence and an incomplete sequence fragments present in the NCBI genome database. Wherein three completed by the sequencing of this laboratory:the standard strains ATCC11845, RA-CH-1(serotype Type Ⅰ) and RA-CH-2(serotype Type II). Due to the scarcity of genome sequence data, the structure of the genome Riemerella Anatipestifer study more scarce, therefore, through our research, we hope to be able to provide basis for Riemerella Anatipestifer research.Isolated from clinical the two Riemerella Anatipestifer (RA-CH-1and RA-CH-2), and buy ATCC11845. Then we have three Riemerella Anatipestifer sequencing and annotation. We conducted a comparative analysis of ATCC11845, RA-CH-1, RA-CH-2and RA-GD in the NCBI database. The analysis results show that the ATCC11845, RA-GD and RA-CH-2three genomes only have a little difference in number of genes, GC content of about35%. RA-CH-1appeared relatively large differences. Genome length is more than three out of140,000bp. RA-CH-1GC content is also slightly higher than the other strains, and the gene number more than100. With the difference rather than the different serotypes of the RA-CH-2greater than the same serotype RA-GD.Riemerella Anatipestifer contains a large number of functional proteins. Mainly the following:Iron transport protein TonB family. The main function of this family is the iron-transport system of the bacteria. Iron absorption is a critical part of the growth of bacteria and iron also has a great influence on bacterial virulence factors, iron transport pathway is very important for maintaining bacterial survival. The capsular exopolysaccharide is important pathogenic factor for many bacteria to the class protein not only be able to suppress many bacterial biofilm formations, but also has the characteristics of the destruction that has been generated biofilms. And in some bacteria there to enhance the effects of some antibiotics destroy the cell’s outer membrane. Two types of efflux systems in Riemerella Anatipestifer genome, which is a Small Multidrug Resistant protein (SMR) family, the other is major facilitator super family (MFS-1). In addition to the above two active efflux system composed of protein, there are still several other antibiotic resistance genes, for example:vanz family proteins, the main function for the vancomycin resistance gene; the penicillinase repressor; acriflavin resistance protein; camphor resistance protein crcb; multiple antibiotic resistances (marc)-related protein.The repetitive sequence is a nucleotide sequence repeated in the genome. These sequences are generally does not encode a polypeptide; can be divided into the interspersed repetitive sequences and tandem repeat sequence. The interspersed repetitive sequences are also known transposable element. Include three kinds:the long terminal repeated, the short interspersed nuclear elements, the long interspersed nuclear elements. The main functions of the long terminal repeat (LTR) are regulating the transcription of viral genes, replication, and integration of the viral gene. Short interspersed nuclear elements (SINE) and long interspersed nuclear elements (LINE) widespread in the eukaryotic genome and occupy a large portion. They are in the form of retrotrans-position inserted into the genome chromosomal new location. They are considered to study the species taxa phylogenetic relationship and population genetics excellent mark, because retrotrans-position are irreversible and independence. Repeat analysis results show that, the four Riemerella Anatipestifers tandem repeat sequence both in copy number or repeat a consistent sequence unit is highly consistent. We do enrichment analysis of between the repeat sequence region and gene overlap region COG function. The results show that, ATCC-11845, RA-CH-2, RA-the GD of the repeat sequence gene, in the folded, recombinant and modified aspects of the function significantly with significant enrichment, while in other functions, did not appear significant enrichment (significant level=0.05). Strain RA-CH-1all the functionality demonstrated significant enrichment.Bacteriophage is a virus infection of microorganisms such as bacteria, fungi, actinomycetes and spirochetes. Bacteriophage integrated in the genome of host bacteria is called prophage. According to the results of previous studies, the prophage in the growth and reproduction of bacteria and the evolutionary process plays an important role. And containing a prophage in the bacterial chromosome is a very common phenomenon. The prophage integration can increase the chance of bacteria obtain foreign useful sequence, enhanced pathogenicity, or to improve their ability to adapt to the living environment. However, the insertion of the prophage may cause the bacteria are cleaved death. By our detection, four genomes failed to identify the presence of a complete prophage. only some fragments of sequence similarity with the phage or prophage.Genomic Island (GI) is the lateral origin evidence characteristic part of the genome. Depending on the kind of carrying the gene, but also can be divided into the pathogenicity island (PAls), the xenobiotic degradation Islands, the metabolic Island, the resistance Island, fitness Islands. Genomic Island has a symbiotic or pathogenic mechanisms or biocompatibility clustering of gene collection, and they have specific structural features. The GC content, nucleotide bias, and the codon usage of the Genomic Island with respect to the level of the chromosome will exhibit differences. The many genes Island also contains part of the exogenous gene, for example, phages and plasmids movable element, can be achieved to parallel move use the self-splicing to other suitable bacteria. This capability is part of the virulence genes or resistance gene is one important way to move between different bacteria, one of the reasons is caused by bacterial multi-drug resistant. Our results show that, RA-CH-1has10genes Island, but have not been able to find similar Genomic Island in the other three Riemerella Anatipestifers. Have a very high similarity to the DNA sequence of these two genes Islands RGL4and RGL9with Ornithobacterium rhinolracheale DSM15997DNA sequence. ATCC11845. RA-GD and RA-CH-2three genomic island between high similarity. Each strain has unique genetic Island.The genes in genes family have a common ancestor, after multiplication and mutation, to form a collection of genes. From the results of analysis of the gene family as well as homologous gene, RA-CH-1has the most distinctive gene family, and up to16, while the other three is only1-2. The630gene familys are the four Riemerella Anatipestifer have, these gene familys account for about half of the number of all the gene familys.This shows that the gene family types of Riemerella Anatipestifers have high commonality; their complexity of the gene family may have an impact on the biological characteristics.We were the four Riemerella Anatipestifer build a phylogenetic tree. At the same time, the conservative element and non-conservative element also constructed a phylogenetic tree. Found through analysis of the conservative element and the non-conservative element topology is consistent, but the branch lengths are not the same. The phylogenetic tree analysis showed strain RA-CH-1, RA-CH-2and ATCC in the same branch nearest. RA-GD belongs to another branch. The analysis showed that of the non-conserved regions and conserved regions, the Riemerella Anatipestifer of non-conserved regions have a higher mutation rate, the mutation rate is about8times the conserved region.更多还原