| Aquaculture industry is an important support to our country economics which develops very fast in recent years. Total aquaculture production of China has got the first place in the world. Aquaculture industry brings us huge economic benefits and remits food pressure to some extent. In the long run, developing aquaculture is not only the aim of China, but also the whole world's significant strategic decision. However, with the transition from extensive to intensive cultivation, the loss caused by many pathogens is rising. Using all sorts of antibiotics accelerated the appearance of resistant strains and brings much more difficulties to disease control. This research preliminarily studied 2 major pathogens from gene function viewpoint--Vibrio harveyi and Edwardsiella tarda.Previous studies revealed that the hemolysin activity in the extracellular product (ECP) of Vibrio harveyi was involved in the pathogenesis in salmonids. The most pathogenic isolate, VIB 645, contained two closely related hemolysin genes (designated vhhA and vhhB).Others contain only one copy or none. These two genes proved to be located in the genome and a plasmid (called pVH1) which is 65.6 kb in size.The open reading frames (ORFs) of vhhA and vhhB shared a high level of identity (98.8%) and were predicted to encode identical polypeptides. Amino acid sequence of VHH shows a high degree of identity (85.6%) to the tlh ( thermolabile hemolysin ) of Vibrio parahaemolyticus.In this study, alkaline lysis method was used to extract the virulence-related plasmid pVH1 from Vibrio harveyi VIB 645. The plasmid was digested by several restriction enzymes (including BglII, Hind III, MboI, SacI, NcoI and PstI) and sequenced by constructing random DNA library using pUC19c as a vector. The total sequence fragments is 28, and 97.63 kb in size, which covered the plasmid 148.3%. 125 ORFs (>150bp) was identified, among which 79 ORFs show high degree of identity to the known data in the GenBank database, 37 ORFs show low degree of identity, and 9 ORFs show no identity which are considered to be potential new genes. These ORFs could be grouped into 5 categories: (1) virulence-related genes, including genes encoding RTX toxins and related Ca2+-binding protein, transferrin-binding protein A precursor, and outer membrane adhesion protein PilT. These genes are related to Ca2+ binding to RTX toxin, iron-uptake system and colonization of pathogens; (2) conjugal transfer related genes, including TraC,-D,-F,-G,-H,-I,-K,-N,-U,-W,TrbB,-C. These genes maybe contribute to the strengthening of viability and virulence of pathogens;(3) transposition related genes; (4) genes with other functions, including methyltransferase, DNA-binding protein RDGA, nitrite extrusion protein, transthyretin, lipoprotein, peptidase, DNA replication protein DnaC and so on; (5) genes with unknown functions.Most of the fragments show high degree of identity to pR99 of Vibrio vulnificus CECT4999, pC4602-2 of Vibrio vulnificus CECT4602 and pYJ016 of Vibrio vulnificus YJ016.So far, there is not clear and minute research about the pathogenic mechanism of Edwardsiella tarda, many virulent factors remain to be found. In this experiment, we chose a PLD superfamily protein which encoded in the genome of standard strain EIB 202 for preliminary research. This gene is 1245 bp in size, encoding 414 amino acids. The 137th and 344th amino acids are the conservative regions of HKD which belongs to the common feature of PLD superfamily protein. With the help of suicide plasmid pRE118, we insert the delete fragment which is homologous to the genome sequence into the genome. Then using screen pressure, we select the specific gene mutant which finished the recombination. Virulence and phenotypic characters are tested. The results show the PLD superfamily protein is not influential to the virulence, LD50 of the mutant and EIB 202 are 2.48×10~4cfu/fish and 2.06×10~4cfu/fish. Phenotypic characters show no significant differences, either. This gene's function remains to be seen. |
