【作者】 谢海侠；

【导师】 聂品；

【作者基本信息】 中国科学院研究生院（水生生物研究所） ， 水生生物学， 2005， 博士

【摘要】 柱形病是一种全球性的危害极广的细菌性病,其病原为黄杆菌科的柱状黄杆菌(Flabobacterium columnare)。柱形病主要危害鱼类的鳃组织,也可引起体表溃乱。细菌性烂鳃病在我国流行较广且危害严重,是草鱼(Ctenopharyngdon idellus)、鳜(Siniperca chuatsi)等重要养殖鱼类的主要病原。本研究以我国分离的引起柱形病的病原为对象,开展了柱状黄杆菌的毒力因子基因的鉴定方面的研究,并对其中的一些毒力因子的功能开展了研究,为最终研制弱毒苗奠定基础。通过对16S rDNA和16S-23S rDNA基因间隔区序列的研究,对从草鱼鱼种上分离的,当时被定名为鱼害粘球菌(Myxococcus piscicola Lu, Nie & Ko, 1975)的细菌性病原的进化关系进行订正,系统树关系表明鱼害粘球菌与已报道的柱状黄杆菌以100%的支持率和极小的遗传变异(对于16S rDNA,为0.8%,而16S-23S rDNA为0.0%)聚为一支,表明鱼害粘球菌为柱状黄杆菌的同物异名。构建了柱状黄杆菌的三个平行表达文库,制备了抗其外膜蛋白(OMPs)的兔抗。对文库进行影印后进行菌落原位杂交,挑取阳性克隆,测序分析,采用基因组步移的方法进行全长克隆,获得4类基因:滑动相关基因、血清抗性相关基因、膜相关的蛋白酶类及胞外胶原酶及蛋白酶基因。此外,通过简并引物获得了柱状黄杆菌的降解多糖的酶-硫酸软骨素AC裂解酶。对滑动基因gldH与gldJ这两个基因进行原核表达,制备兔抗,然后将柱状黄杆菌通过超速离心进行细胞分相,通过Western-blotting,将GldH及GldJ定位在细菌的外膜相。此外,借助同源双交换,缺失滑动基因gldH内部约300 bp片段并定向插入抗性筛选标记,获得突变子,对突变子的菌落形态的改变、细胞壁荚膜多糖层变化、滑动能力及粘附特性进行了分析,发现突变子菌落边缘假根消失,菌落变得很平滑; 荚膜多糖层变薄; 滑动能力丢失; 粘附能力只有野生型的约1/4。通过设计简并引物,获得了硫酸软骨素AC裂解酶的基因片段,然后通过基因组步移方法,获得了其全长。对其全长进行原核表达,在非变性条件下纯化重组硫酸软骨素AC裂解酶,并将硫酸软骨素A加入到柱状黄杆菌培养基中诱导天更多还原

【Abstract】 Columnaris is a world-wide bacterial fish disease, causing heavy economical losses in aquaculture industry. The bacterial pathogen, Flavobacterium columnare belongs to the family Flavobacteriaceae. The infection of the bacterium may cause severe gill-rotting and sometimes skin ulceration. Bacterial gill-rot disease is epidemic in China, and a major disease in several major cultured fish species in China, including the grass carp Ctenopharyngodoe idellus and the mandarin fish Siniperca chuatsi. The present study, by using bacteria isolated from outbreaks of columnaris disease in China as the pathogen, aimed to identify genes of possible virulence factors of the bacteria, and functions of some of these identified genes were also examined. The bacterium causing columnaris disease was identified first as Myxococcus piscicola Lu, Nie & Ko, 1975 from diseased grass carp in China. In order to clarify the identity of this bacterium, 16S rDNA and 16S-23S rDNA intergenic spacer region sequences were cloned from the so-called M. piscicola. As revealed by the high percentage of boostrap values with other species in the genus Flavobacterium and other strains of F. columnare isolated in other countries, the bacterium, M. piscicola causing columnaris disease isolated from grass carp should be a synonym of Flavobacterium columnare, and the most virulent strain isolated from grass carp, i.e. the G₄ strain was employed in the present study. In order to identify genes encoding the outer membrane proteins （OMPs） of F. columnare G₄, the expression library of the bacterium was screened by using rabbit antisera developed against its OMPs. Positive colonies of Escherichia coli M15 containing fragments encoding the bacterial OMPs were selected for cloning the relevant genes by genomic walking methods. A total of 36 genes were obtained, and 11 of which are possible virulence factors which can be divided into four categories: 1) gliding related genes, 2) genes related to eliminating the killing ability of the host, 3) membrane-associated proteases, and 4) extracellular protease and collagenase. The gliding related gene gldH and its role in gliding motility, chitin utilization and adherence was examined in F. columnare G4. Western blotting analysis using rabbit antisera against recombinant GldH showed that the protein is localized in the outer membrane fraction of the bacteria. The defined deletion of the chromosomal copy of gldH in wild-type F. columnare G4 has led to the establishment of a gldH deficient mutant. Functional experiments have shown that the gldH mutant loses its abilities in cell gliding, colony spreading, chitin digestion, and adherence to gill tissues. The mutant obtained from defined gene deletion in the present study represents the first description of an adhesion-defective mutant for F. columnare, and the techniques involved in the present study may provide basis for further study on other genes of F. columnare and also other Gram-negative bacterial pathogens. Gene and functional analysis were also conducted for the glycosaminoglycan-degrading enzyme chondroitin AC lyase from F. columnare G4. The chondroitin AC lyase gene was obtained by degenerated primers and then genomic walking methods. The gene, cslA was then successfully expressed in E. coli and recombinant chondroitin AC lyase, rChonAC was purified, with the lytic activity of rChonAC analyzed. Zymography analysis copolymerized with chondroitin sulphate revealed the lytic activity of rChonAC and also the crude native ChonAC isolated from periplamic space of cultured F. columnare G4. The low level of lytic activity as observed in crude native ChonAC may be due possibly to the low level of expression of this gene in the cultured condition. The expression and the role of this virulence factor is of interest for further research on the pathogenesis of F. columnare. In addition, histological and cytological studies on the developing thymus and pronephros of the mandarin fish Siniperca chuatsi （Perciformes: Teleostei） were also conducted in the present study. The thymus of the mandarin fish was examined by light and transmission electron microscopy to understand its formation and cellular composition. Obvious thymus was found packed with lymphocytes on 7 dph. The thymus of S. chuatsi was connected with the extension of the third, fourth and fifth branchial pouches throughout the early development, and remained its superficial position in the adults, with perforations observed on its surface. In thymus of the adultfish, thymic epithelial cells （TECs） characteric of tonofilaments were observed, with limiting TECs （LECs） found in subcapsular, subseptal, perivascular, and nurse-like TECs containing viable intact lymphocytes inside their vacuoles. In addition, three kinds of granulocytes were observed throughout the thymus, and incomplete blood-thymus barrier found in the inner zone. Taken together, thymus development in mandarin fish agrees, to some extent, with ontogenetic patterns observed in other fish species. The renal tubules and lymphoblast-like cells could be observed one day after hatching. And the developing process could be subjectively divided into three stages. At the first stage, only renal tubules could be observed, and the pronephros functions as urinary organ; with pronephros development, more lymphoid cell appears, and the pronephros seems bifunctional, i.e. it functions as an urinary organ and a lymphoid organ as well; then with it comes to 35 dph, the number of the renal tubule decreased rapidly, and the pronephros is occupied completely by lymphoid cells. In summary, possible virulence genes of F. columnare G4 have been screened successfully by using antisera developed against the OMPs of the bacterium, and a total of 15 such genes have been sequenced. Techniques for defined gene knock-out have been established for understanding the function of the possible genes. The present study thus provide a foundation for further investigation on the virulence factors of F. columnare G4 and their interactions with fish hosts.更多还原