Research On Novel Detection Methods For Pathogen And Phenoloxidase Pathway Genes In Fenneropenaeus Chinensis

Shrimp cultivation has been suffering great losses due to the outbreak of diseases.To develop rapid detection of aquatic animal diseases and to take quatantine measures in time to control disease were equally important,in which the key step is to develop rapid detection method and to further reveal immune mechanism and find out effective method to control shrimp disease.Researches showed that when shrimp and other crustaceans were stimulated by outside pathogens,ProPO system in crustaceans would be activated a very small amount of microbial polysaccharides.A series of active substances,such as melanoma, prophenoloxidase activator factor(PPA),pattern recognition protein(BGBP,PGBP, LGBP,LBP)and membrane Giant globulin receptor and A2 were involved in the activation process.All these proteins participate in defense response,including the provision of conditioning,and promote blood cell phagocytosis,the formation of cysts and nodules or lectin-mediated and solidification,melanin production. Melanoma form black dot on the arthropod surface,which plays a role of body protector.Therefore,the reaction in prophenoloxidase path way is the key factor for the immune cascade arthropod.In the thesis,the loop-mediated isothermal amplification(LAMP)method for rapidly detecting white spot syndrome virus species and V.anguillarum in shrimp were found.And two key genes of phenoloxidase system(prophenoloxidase and Serine protease FCSP3)related to the immune system in Chinese shrimp F.chinensis have been clone and analyzed.Fragments of the VP28 gene of the WSSV virus and empA gene of V. anguillarum could be individually amplified at 65℃in the presence of a primer mixture and Bst DNA polymerase.Amplification products were detected by visual inspection and agarose gel electrophoresis.The specificity of WSSV LAMP primers was demonstrated by the absence of amplification of DNA from healthy shrimp,and specificity of V.anguillarum LAMP primers was confirmed by ten non- V. anguillarum Bacteria.Compared with the performance of the established PCR and LAMP assay,both two developed LAMP methods had appeared to have wider spectrum for WSSV virus and V.anguillarum in detection.The prophenoloxidase(ProPO)gene was cloned from haemocytes of Chinese shrimp F.chinensis by Rapid Amplification Complementary DNA Ends(RACE) method.The full-length cDNA of ProPO consists of 3040 bp with a 2061 bp Open Reading Frame(ORF),which encodes 686 amino acids,and a predicted signal peptide of 12 amino acids.It showed 93%and 92%homology with those of P. monodon and P.semisulcatus.To understand its function,amounts of ProPO expressions in different tissues were studied by real-time PCR after challenged by V. anguillarum.The results showed that the expression level of ProPO in haemocytes was highest among three studied tissues which were haemocytes,hepatopancreas and lymphoid organ.After V.anguillarum injection,ProPO in haemocytes was observed at a lower mRNA level was observed at 6 hour post injection(hpi) compared with that of control,and then increased.The level graduately grow until it reached the peak at 12 hpi.In lymphoid organ of shrimp,the ProPO level was up-regulated to four times as normal level at 6 hpi,followed by a down-regulation. In all three organizations tissues,the controls group had changes less than the infected group,and the differences were significant.According to EST of hepatopancreas cDNA library of F.chinensis,a serine protease was cloned using RACE method.The full-length cDNA of Serine protease FCSP3 consists of 1622 bp with a 1431 bp ORF,which encodes 477 amino acids and a signal peptide of 22 amino acids.This gene show a 33%homology with Serine protease of A.gambiae,and 32%homology with prophenoloxidase activator factor of N.vitripennis,34%homology with prophenoloxidase activator factor of H. diomphalia.The expression of serine protease was highest in the lymphoid organs, was lowest in haemocytes,The expression of FcSP3 in Lymphoid s after V. anguillarum injection increased significantly from 12 hpi to 72 hpi,while the control group changed a little.In lymphoid organ of shrimp,the FcSP3 level was down-regulated to 1/10000 times as normal level at 6 hpi,followed a sustaining low level.In hepatopancreas of shrimp,the FCSP3 level increased remarkable to 1.3 times as normal level,then droped back to normal at 24 hpi.