The Effects Of MyD88 In The Process Of Development And Immune In The Haliotis Divercolor

Haliotis diversicolor is an important shellfish,playing an important role in the world.For the reason of disease in Haliotis diversicolor caused mass mortality of abalone,the study of abalone growth and immune mechanism is essential.MyD88 is a classical important player in TLR signaling pathway activating NF-κb and apoptosis signal pathway which plays an important role in the immune system.Meanwhile,MyD88 is an important member in Toll-dorsal differentiation pathways which plays an important role in growth mechanism.Partial sequence of two MyD88 were obtained from our EST database of small abalone,and the full-length cDNA sequences of the two genes were cloned by SMART RACE techniques,which were denoted hdMyD88 and hdMyD88-2.Real-time quantitative PCR(RT-qPCR),RNA interference(RNAi)and in situ hybridization were applied to reveal the role of development and immune of two MyD88.The main results of this study were reported as follows:1)The full-length cDNA of hdMyD88 is 1927 bp,and the open reading frame encodes a protein of 438 amino acids.The predicted protein contains a MyD88 DD domain(215-460 bp)and TIR-2 superfamily domain(665-1000 bp).DD domain contains a MyD88-IRAK4 interaction sites.The predicting protein molecular weights about 48.92 kDa and isoelectric point of approximately 5.63,without signal peptides,disulfide bonds and transmembrane domain.The predicting protein has 1 N-glycosylation sites,46 serine phosphorylation site,6 threonines and 5 tyrosines.The full-length cDNA of hdMyD88-2 is 1820 bp,and the open reading frame encodes a protein of 350 amino acids.The predicted protein contains a MyD88 DD domain(247-475 bp)and TIR-2 superfamily domain(823-1228 bp).The predicting protein molecular weights about 39.65 kDa and isoelectric point of approximately 8.05,without signal peptides,disulfide bonds and transmembrane domain.The predicting protein has 4 N-glycosylation sites,14 serine phosphorylation site,7 threonines and 2 tyrosines.2)In the process of embryos and larvae development,from eggs,two cell,eight cell to morula period,the expression of hdMyD88 is stable;from 128 cell the expression of hdMyD88 is gradually declined;from morula stage to the late veliger stage,the expression of hdMyD88 are lower when compared with 8 cell and early creep are very significantly differences(p <0.01).The expression of IRAK4 from eggs to two cells is relatively stable,and the expression of IRAK4 in eight-cell stage increased dramatically,then expression in following shage decline and has a stable expression.The expression of IRAK4 in eight cell is high significantly sex differences(p <0.05).The expression of AP1 in segmentation,embryo development stage and veliger is stable and lower compared with early creep(p <0.05).The expression of TNFα from egg to 128 cell gradually declined,but no significantly sex differences(p >0.05);then expression gradually rise,in early creeping.The expression of CAT from the egg until the middle veligeris a relatively stable compared with the early creeping(p <0.05).The expression between SOD and CAT were similar.The expression of MyD88-2 from eggs until the trochophore larvae is at a lower level(p <0.05),then decreased in mid veliger and rise in early creep(p <0.05).3)Trochophore larvae and early veliger were immersed with dsMyd88 and dsEGFP as negative control.Among the dsMyD88 group,blank group and negative group,there are no significant difference in survival(p >0.05).Results show that the expression of Myd88 is significantly different between dsMyD88 and negative control group in the trochophore larvae based on different internal parameters(p <0.05).The expression of IRAK4 which is the downstream gene of MyD88 in Toll signal pathway obviously declined(p <0.05).The expression of AP1 significantly increased(p <0.05).The expression of TNF alpha significantly increased(p <0.01).The expression of CAT of expression obviously declined(p <0.05),and the expression of SOD has no differences(p >0.05).The expression of MyD88-2 is lower than the blank Group(p <0.05).Results show that the expression of Myd88 is significantly different in the early veliger larvae between dsMyD88 and negative control group based on different internal parameters(p <0.05).And the expression IRAK4 which is the downstream gene of MyD88 in Toll signal pathway obviously declined(p <0.05).The expression of AP1 and TNFα increased(p <0.01).The expression of CAT of expression has no differences(p >0.05),and the expression of SOD has differences when compared with positive group and negative group(p >0.05).The expression of MyD88-2 has no differences(p >0.05).4)MyD88 was detected in ventral specificlly from early trochophore larvae to early veliger larvae by the means of in situ hybridization in positive control group and negative control group.The signal cannot be detected in larvae in RNAi interference group after 6 hours.When adding Vibrio parahaemolyticus in RNAi group,there is weak signal can be detected.5)The condition of primary cell culture was optimized in Haliotis diversicolor and these primary cells of hemocyte and gill were used in RNA interference(RNAi)and exogenous gene expression.Results suggested that hemocyte survived better in higher osmolarity of media,the way of using lymphocyte separation medium to culture hemocyte was beneficial for cell growth.MyD88 dsRNA were added to cell culture medium of hemocyte and gill after 5 days of culture which reduced the expression of MyD88.The plasmid pEGFP-N1 was transfected into gill after 5 days of culture and the green fluorescence was detected 5 days later.Above all,the primary cell can survived in vitro and they are a proper tool for gene function analysis by regulating gene expression up or down,which provide convenient way for functional annotation.