Pathological Study Of Several Pathogenic Bacteria Infected With Octopus Ocellatus And The Cloning And Expression Of TLR13 Gene

O.ocellatus is an economic species in northern coastal areas of China.In recent years,as the environment has deteriorated and O.ocellatus has been overfished that led to a shortage of O.ocellatus,artificial culture of O.ocellatus is urgent.The bacterial disease caused by pathogen infection in the process of aquaculture has brought a lot of problems and challenges to the aquaculture industry.In this study,the histopathological findings of O.ocellatus infected with vibrio anguillae and vibrio parahaemolyticus were studied;The changes in the structure and diversity of O.ocellatus liver after pathogen infection were detected by 16 S amplification technique;Finally,the expression patterns of TLR13 gene in different tissues of O.ocellatus and at different time points after infection with vibrio anguillaris were analyzed,In order to provide the theoretical basis for the prevention and control of O.ocellatus and the immune recognition molecular response mechanism.Specific research contents are as follows:(1)From a histopathological point of view,O.ocellatus artificially infected with vibrio anguillae and vibrio parahaemolyticus was studied,histological sections showed that ameloctopus had obvious pathological changes in liver,gill heart,gill and white body.The liver cells present many vacuoles,membrane splitting,the structure of the liver locking scaffold also became incomplete;In addition to obvious morphological changes in gill cells,The gill lobule and epithelial microvilli were damaged,these will affect the gill respiration,excretion and other functions;The white body contains a large number of hematopoietic stem cells,progenitor cells and blood cell precursor cells and blood cells,The white body is also the body's good blood supply,In addition to the obvious vacuolization,there were some cell fragments and cell shrinkage which resultedin the decrease of hematopoietic function.The phenomenon of nuclear membrane rupture and nucleolus disintegration occurred in gill heart cells,the function of removing bacteria and detoxification of gill heart was decreased.According to the sparsity curve and species abundance clustering tree,The difference between vibrio anguillae infection group and control group was obvious,The structure and diversity of the Edwardian group were more similar to that of the control group.(2)The structure and diversity changes of O.ocellatus liver after infection with vibrio anguillae and Edouardian were analyzed by 16 S amplification technique.The results were as follows: Shannon index in vibrio anguleae infection group was <edwardsian infection group < control group,while Simpson index in vibrio anguleae infection group was > edwardsian infection group>control group,The results showed that the infection of vibrio anguleae and Edouardian caused the decrease of community diversity,The community diversity of Edwardian infection group was higher than that of vibrio anguillae infection group.The above results indicated that vibrio anguillus had a stronger effect on octopus amoctopus liver.(3)The full-length c DNA of TLR13 2608 bp,containing a 5' terminal un-translateed region(UTR)of 201 bp,343 bp's 3 'UTR,2064 bp open reading frame(ORF),a total of 668 amino acids were encoded,Its molecular weight was predicted to be 79.5KDa,The theoretical isoelectric point is 8.83.Amino acid structure was predicted by SMART,The sequence consists of a signal peptide and three domains.TLR13 m RNA was detected by real-time fluorescence quantitative PCR in healthy O.octopus liver,gill,white body,gill heart,muscle and blood cells,the expression in the white body was the highest,and the expression in the liver was the lowest.Vibrio anguillae infection with octopus can cause up-regulation of TLR13 m RNA in liver.The peak was reached 24 h after infection,that's 6.97 times higher than before the stimulus.The increase of TLR13 suggests that this gene may be involved in the immune response against invasion by exogenous pathogens.From what has been discussed above,pathological observation of O.octopus infected by vibrio anguillus and vibrio parahaemolyticus was carried out,It was found that all the tissues in had O.ocellatus obvious pathological changes.The structure of O.ocellatus liver was analyzed by 16 S amplification technique,to compare the changes of O.ocellatus liver flora after vibrio anguillae and Edwardian infection.The TLR13 gene of O.ocellatus was cloned.Its expression in each organization was tested,and theexpression pattern of vibrio anguillae at different time points after infection.It was found that vibrio anguillus could induce the up-regulation of TLR13 gene in O.ocellatus,It shows that O.ocellatus TLR13 as a pattern recognition receptor,vibrio anguillae can be identified.The above results indicated that the characteristic changes of the activity,the mortality and the pathological changes of the immune-related tissues and organs of O.ocellatus after pathogen infection.It is of great significance for disease prevention and control and accurate diagnosis in future large-scale culture of octopus octopus.