Study On Pathogen Of Skin Ulceration Syndrome And Construction Of SSH CDNA Library Induced By Vibrio Pomeroyi (Thompson,2003) With Analysis Of Expressed Sequence Tags In Saestival Apostichopus Japonicus (Selenka,1867)

The sea cucumber,Aposrtichopusjaponicus(Selenka,1867),is a major aquacultural species along the coast of northern China.Because of increasing demand of the profitable species,culture of A.japonicus has expanded to the southern region of Chinaincluding Fujian,Guangdong Provinces.However,as a temperate species,A.japonicus is more easily infected by environmental bacteria during aestivation,and skin ulceration syndrome is proved to be the major disease of sea cucumber.In the southern region of China,relative high temperature in the water column reduces the immunity of the species and facilitates burst of the diseases.Massive mortality of causing by the disease does a great damage to the aquaculture business in Southern China.Accordingly,this study has defined the pathogens of diseased sea cucumber with skin ulceration syndrome,and established rapid diagnosistechniques.By applying the molecular biological techniques,immune-related genes have been isolated from sea cucumber induced by bacterial pathogens which provide evidence to reveal the immune defense mechanism.Differences in gene expression of diseased A.japonicushave been analyzed to evaluate the relationship between stress from bacterial pathogens of skin ulceration syndrome and immune relative gene.This study provides fundamental knowledge of the mechanism of the immune defense system in A.japonicusand sheds light on cultivating disease-resistant species.The main results are as bellow:1.In this study,a dominant bacterium strainis isolated from the body wall of diseased sea cucumber(A.japonicus)infected with skin ulceration syndrome.Meanwhile,the artificial infection test proves that V.pomeroyican cause severe infections and even death whether injection or immersion.Phenotypic characterization of the bacteria is investigated with traditional biochemical tests.Genotypic characterization is evaluated by 16S rRNA gene sequencing technique,along with matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS).The phenotypic and genotypic characterizations prove that the pathogen is Vibrio pomeroyi.MALDI-TOF MS analysis is proved to be an accurate and rapid method for identification of common pathogens.2.V.pomeroyi SUS isolated from China with two other strains of V.pomeroyi,including LMG20537T(type strain)isolated from Brazil and V.pomeroyi929 isolated from the GreatPeterBay,Sea of Japan have been compared.And the result shows that there is distinctly different tolerance pattern with environmental factors such as salinity and temperature.Unlike the strain V.pomeroyi SUS,the strains V.pomeroyi LMG20537T andV pomeroyi 929 didn't grow at 35?.Moreover,V.pomeroyi LMG20537Tadapted to the medium with 8%NaCl,but others had no growth.3.Suppression subtractive cDNA hybridization(SSH)is used to identify immune-relevant genes in response to challenge with Vibrio pomeroyi.Subtractive cDNA libraries are constructed from coelomocytes of sea cucumber during the infection process.Approximately 800 clones have been sequenced,giving a total of 318 non-redundant ESTsin response to V.pomeroyi challenge.Using the gene ontology(GO)classification,I successfully assign functional annotations to 318 of the unigene sequences.The number of terms of biological process is the most,the number of terms of cellular component and molecular functionare approximate.In the GO cellular component ontology,the largest proportion of unigene is cell part;in the GO molecular function ontology,the most commonly occurring terms are protein binding;and in the GO biological process ontology,regulation of biological process is the terms that occurred most frequently.4.As a result,5 immune-related ESTs are selected and studied to compare the immune response of A.japonicus following Vibrio pomeroyi infection.The five ESTs belong to C-type lectin(contig116 and contigl7),iron-containing protein(B07 and HO1)and serum amyloid A protein(SAA)(contig96)respectively.Expression levels of 5 immune-related ESTs in coelomocytes from sea cucumber at 24 h and 48 h along with the expression of each EST in different organs/tissue are determined by quantitative real-time PCR to verify their differential expression after V.pomeroyi challenge.The results show that the temporal expression patterns of contig116 and contigl7 are different over time after challenge due to the different types of C-type lectin;but expression patterns of two ESTs in different organs/tissue are relatively consistent.The highest expression is in coelomocytes,and the lowest expression is found in the body wall,contig17 even is not expressed in the body wall.Comparing to the control group,the expression of serum amyloid A increase at 24h after injection,and it is significantly lower at 48h after infection.Therefore,these data suggested that A.japonicus has no acute-phase response at 48h after injection according to the characteristics of this protein.Iron-containing protein including the ferritin(B07)and the ribonucleotide reductase(H01)in this study.After injection,the highest expression of the ferritin is in the respiratory tree,and the highest expression of the ribonucleotide reductase is in the coelomocytes,the secondary is in the respiratory tree.The expression patterns of the ferritin and the ribonucleotide reductase are different at 24h after induce,the former is down-regulated,but the latter is up-regulated.The iron-containing protein isrelated to not only oxygen transport and electron transfer functions,but also immune defense functions.