Transcriptome Analysis Of Genes And Signaling Pathways Involved In Immunity Under Thermal/hypoxic Stresses In Haliotis Diversicolor

The small abalone Haliotis diversicolor belongs to Mollusca,Gastropoda,Prosobranchia,Archaeogastropoda,Haliotidae,Haliotis.It is one of the most commercially important cultured abalones of coastal provinces of southern China.With the increasing of aquaculture density and the rising of temperature,the thermal and hypoxia stresses have become a serious problem.When the small abalones suffer dual/multiple stresses,the normal physiological state will be affected and led to immunosuppression.That will be a negative impact on small abalone aquaculture.In this study,in order to understand the mechanism of immunosuppression when small abalone has been suffered from hypoxia stress and bacterial challenge,the transcriptomes of H.diversicolor haemocytes involved in immunity under stresses and normal conditions were profiled by using IIlumina Hiseq TM2000 to identify genes and signaling pathways that are related to immunosuppression.Additionally,some important immune related genes and signaling pathways were chosen to analyze their expression.Meanwhile,PI3K-AKT signal transduction pathway as an evolutionarily conserved innate immune pathway which plays a critical role to resist external stimuli from invertebrate to vertebrate and was another topic of this research.The results are shown as follows:1.307395572 clean reads generated fromRNA-seq were assembled into 99774 unigenes(mean size=768 bp)from 14 blood samples of small abalone under hypoxic stresses.Based on sequence similarity searches,47154 unigenes were assigned to NCBI non-redundant protein sequences(Nr)which provides massive data for excavating immune-related genes and signaling pathways.KEGG analysis showed that 242 unigenes annotated with immunologic function were mapped into several immune-related pathways,including MAPK signaling pathway,P38 signaling pathway,NF-?b signaling pathway,HIF signaling pathway,and PI3 KAKT signaling pathway.Furthermore,24189 differential expression genes(DEGs)were found,14271 DEGs(59%)were annotated with known protein sequences from the Nr protein database.Finally,we chose 41 unigenes to verify the results of transcriptome analysis(RPKM)by quantitative real-time PCR(q RT-PCR),and expression leve of 21 unigenes measured by q RT-PCR was consistent with the results of transcriptome analysis.2.The PI3K-AKT signal pathway has been found to be involved in many important physiological and pathological processes of innate immune system of both vertebrates and invertebrates.In this study,AKT(Hd AKT)and PI3K(Hd PI3K)gene of small abalone H.diversicolor were cloned and characterized due to the important status of PI3 K and AKT protein in PI3K-AKT signaling pathway.Hd AKT c DNA is 2126 bp,with an open reading frame(ORF)of 1440 bp encoding a protein of 479 amino acids,a 5'-untranslated region(UTR)of 198 bp,and a 3'-UTR of 488 bp.The length of the complete c DNA sequence of Hd PI3 K is 6052 bp and the c DNA sequence contains an ORF of 3294 bp that encoding for 1097 amino acids,a 5' UTR of 496 bp and a 3'-UTR of 2262 bp.3.The mRNA expression level of fourteen genes of PI3K-AKT signaling pathway were detected by q RT-PCR.The results showed that all these fourteen genes were ubiquitously expressed in seven selected tissues.Meanwhile,Hd AKT was expressed in haemocytes with the highest expression level(p < 0.05)next in hepatopancreas(p < 0.05).On the other hand,the expression level of Hd PI3 K in haemocytes was higher than other tissues.Under normal condition,the gene expression level of Hd AKT,Hd PI3 K,and other PI3K-AKT signaling pathway members were significantly up-regulated by Vibrio parahaemolyticus infection.The results demondtrated that Hd AKT,Hd PI3 K,and other PI3K-AKT signaling pathway members play their roles in innate immune system in abalone.The mRNA expression of these genes in gills,haemocytes,and hepatopancreas was significantly down-regulated after the V.parahaemolyticus stimulation with environment stimulation(thermal,hypoxia and thermal & hypoxia).The results indicated that the dual/multiple stresses defeated immune system and led to immunosuppression in abalone.PI3K-AKT signaling pathway may be involved in hypoxia/thermal-induced immunosuppression of small abalone H.diversicolor.4.When the HdAKT was inhibited by dsRNA,the expression level of HdAKT was lower than the blank control group and GFP group in hemocytes at 4 h,12 h and 24 h(P < 0.05);Meanwhile,the expression levels in most of PI3K-AKT signaling pathway members were significantly changed than the blank control group and GFP group in culture haemocytes(P < 0.05).All these PI3K-AKT signaling pathway genes were established in a molecular network by Cytoscape after the inhibition of Hd AKT.The results indicated that the expression of all PI3K-AKT signal pathway genes was significantly lower than the blank control group at all examined time points when the Hd AKT was inhibited by ds RNA.Resuts of this study showed that Hd AKT play a key role on the regulation of other genes in haemocytes and functions as a positive regulator of members of PI3K-AKT signaling pathway.