| Black bream(Acanthopagrus schlegelii)belongs to the Sparidae of Perciformes.Because of rapid growth,high-nutrition value and meat quality,A.schlegelii is loved by many consumers leading to a broad market.The pattern of A.schlegelii aquaculture mainly consists of cage aquaculture and pond aquaculture in sea water.With the continuous expansion of cultivation scale,the continuous improvement of the intensification degree and the increasingly serious pollution,the number of pathogens in the seawater was increasing and leading to the occurrence of bacterial,parasitic and viral diseases of A.schlegelii more and more frequently.These diseases have caused great economic loss to the breeding production and seriously hindered the development of the breeding industry of A.schlegelii.It's more important to prevention than treatment,so the immunity of A.schlegelii forms a protective umbrella against epidemics and plays a role in preventing diseases.The immunity of fish is very important for reducing drug using and ensuring the healthy development of aquaculture.This study focused on the immune genes Myd88 and I?B? of A.schlegelii by molecular biology techniques and biological information analysis,aiming to explore a series of physiological and biochemical reactions caused by pathogen invasion.It provides a theoretical basis for improving immunity and disease resistance and promoting the development of aquatic industry.This study mainly includes the following aspects:1.Genetic cloning and bioinformatics analysis of Myd88 and I?B? of A.schlegeliiThe ORF of Myd88 in A.schlegelii was 897 bp,encoding 299 amino acids.The molecular weight and the isoelectric point of Myd88 was 34.30 k Da and 5.14,respectively.The ORF of I?B? in A.schlegelii was 948 bp,encoding 316 amino acids.The molecular weight and the isoelectric point of I?B? protein was 35.75 k Da and 4.67,respectively.Myd88 of A.schlegelii had the highest homology with zebrafish Myd88(68.88%),and the homologies with human type 1 Myd88,type 2 Myd88 and mice Myd88 were 59.58%,61.29% and 62.01%,respectively,while the homology with fruit fly Myd88 was lower(28.38%).The highest similarity between zebrafish and A.schlegelii I?B? protein was 53.54%,and its homology with mice I?B? and human I?B? was 48.52% and 49.19%,respectively,while that with fruit flies I?B? was 32.02%.2.Distribution and expression pattern analysises of Myd88 and I?B? genes of A.schlegeliiMyd88 and I?B? genes were expressed in all tissues of healthy A.schlegelii.I?B? is also highly expressed in the blood and less so in muscle and intestine.Myd88 expressions in the heart,gill,kidney and blood were up-regulated after Vibrio parahaemolyticus infections.I?B? expressions in the heart,kidney and blood were down-regulated after Vibrio parahaemolyticus infections,while up-regulated in the gill.The trends of expression in the organization were almost on the contrary.Therefore,Myd88 and I?B? genes play a crucial role in A.schlegelii to resist the pathogen invasion.3.The expressions of downstream inflammatory factor caused by overexpressions of Myd88 and I?B?Mammalian overexpression vector was constructed and transferred into Chinese hamster ovary(CHO)cells,then detect the expressions of IL-1?,IL-6,IL-12 and IL-18.It was found that the overexpressions of Myd88 and I?B? resulted in the downstream inflammatory factors expression changed in different degrees.It can be concluded that Myd88 and I?B? were two key genes,played a role in regulating the expression of downstream inflammatory factors,and then regulating the immune capacity of the body. |
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