Studies On NF-κB Signaling Pathway-related Genes From Haliotis Diversicolor Under Thermal And Hypoxic Stresses

The small abalone Haliotis diversicolor is a marine gastropod mollusk in the family Haliotidae.It is one of the most commercially important cultured abalone of coastal provinces of southern China.However,the deteriorating environmental conditions and infectious diseases have become the major problems that have threatened the abalone aquaculture industry for a long time,especially in summer period.The amount of dissolved oxygen in sea water is diminished as the result of high temperature.In particular,hypoxia caused by the elevated temperature can change metabolic rate and respiratory rate of marine benthic organisms and can result in high mortality of them.In addition,infectious diseases also play a key role in the outbreak of mass mortality of cultured abalone and cause catastrophic losses to aquaculturist.As a result,the defense system of the body will come into play in order to survive under these stress conditions.Due to the lack of the adaptive immune system,abalones only rely on the innate immune system to resist all these stresses.So it is necessary for us to study the innate immune defense mechanism of abalone.Here in our research,we isolated and characterized,for the first time,four genes of NF-κB signal pathway by using degenerate primer PCR amplification,transcriptomic database searching and SMART-RACE technology from H.diversicolor : NF-kappa-B inhibitor(IκB),Akirin2,Tyrosine 3/Tryptophan 5 monooxygenase activation protein zeta(14-3-3ζ),Ras homologenriched in brain(RHEB).They were named as SaIκB,SaAkirin2,Hd14-3-3ζ and HdRHEB.Meanwhile,we predicted and analyzed the domains and structures of these genes by using different bioinformatic methods.Furthermore,we studied the mRNA expression levels of several genes linked to NF-κB signal pathway under thermal stress,hypoxia exposure and thermal plus hypoxia stress by using Real-time PCR.Also,we detected mRNA expression level of other genes of NF-κB signal pathway by using RNAi technology to inhibit SaIκB.The results were as follows:(1)The full length cDNA of SaIκB was 1748 bp encoding 401 amino acid residues.qRT-PCR indicated that SaIκB could be detected in all the examined tissues and it was predominantly expressed in the tissue of gills.The expression of SaIκB in gills was significantly higher at 4 h and 96 h respectively(P<0.05)under hypoxia condition.Meanwhile,it was also up-regulated significantly in haemocytes at 24 h,96 h and 192 h(P<0.05).However,the mRNA expression level showed that there was no significant effect(P< 0.05)of thermal treatment in haemocytes until 96 h.And in gills,the expression of SaIκB was up-regulated significantly(P< 0.05)only at 192 h.Under the thermal plus hypoxia stress,in gills,SaIκB mRNA expression was markedly up-regulated at 4 h and 24 h while in haemocytes the expression level was significantly up-regulated at 4 h only.(2)The full length cDNA of SaAkirin2 was 1452 bp encoding 187 amino acid residues.qRT-PCR indicated that SaAkirin2 could be detected in all the examined tissues and it was expressed at the highest level in hepatopancreas,followed by mantle.Under hypoxia condition,the expression level of SaAkirin2 mRNA in haemocytes was markedly increased at 4 h,96 h and 192 h(P<0.05).Meanwhile,it was significantly up-regulated only at 192 h in gills.After thermal stress,a significant expression change of SaAkirin2 was only detected at 192 h in gills.Meanwhile,SaAkirin2 mRNA expression was significantly up-regulated at 96 h and 192 h in haemocytes.SaAkirin2 expression was up-regulated significantly at 24 h and 192 h posts the thermal plus hypoxia stress of the gills and at 4 h and 96 h respectively in haemocytes(P< 0.05).(3)The full length cDNA of Hd14-3-3ζ was 3010 bp encoding 272 amino acid residues.qRT-PCR indicated that Hd14-3-3ζ was detected in all examined tissues,with the highest level in hepatopancreas,followed by haemocytes.Under hypoxia condition,the expression level of Hd14-3-3ζ in gills was up-regulated significantly only at 4 h(P<0.05)while in haemocytes the significant change was detected at 24 h and 96 h(P<0.05).After thermal stress,the Hd14-3-3ζ mRNA expression level was up-regulated significantly only at 96 h in gills(P< 0.05)while there was no difference in haemocytes.Under the thermal plus hypoxia stress,the Hd14-3-3ζ mRNA expression level was up-regulated significantly at 4 h,24 h and 96 h in gills(P< 0.05)while in haemocytes the expression level of Hd14-3-3ζ was no significant difference between control and exposed groups.(4)The full length cDNA of HdRHEB was 1044 bp encoding 182 amino acid residues.qRT-PCR indicated that HdRHEB could be detected in all the examined tissues and it was expressed at the highest level in mantle(P< 0.05).Under hypoxia condition,the expression level of HdRHEB mRNA in gills was markedly increased at 4 h,24 h and 96 h(P<0.05).Meanwhile,it was significantly up-regulated at 24 h,96 h and 192 h in haemocytes.After thermal stress,a significant down-regulated change of HdRHEB was detected at 4 h and 24 h in gills.Meanwhile HdRHEB mRNA expression was significantly down-regulated all the time in haemocytes.HdRHEB expression was up-regulated significantly at 24 h and 96 h posts the thermal plus hypoxia stress of the gills and it was significant up-regulated all the time between control and exposed groups in haemocytes(P< 0.05).(5)Seventeen other genes linked to NF-κB signal pathway from H.diversicolor EST were obtained.They were Nuclear factor κB(NF-κB),B-cell lymphoma leukemia 10(BCL10),interleukin-1 receptor-associated kinase 4(IRAK4),mitogen-activated protein kinase kinase kinase 7(TAK1),myeloid differentiation primary response protein 88(MyD88),NF-kappa-B inhibitor-interacting Ras-like protein 2(NKIRAS2),nf-kappa-b-repressing factor(NKRF),TNF receptor-associated factor 6(TRAF6),TNF receptor-associated factor 2(TRAF2),toll-like receptor 2(TLR2),toll-like receptor 4(TLR4),toll-like receptor 6(TLR6),thioredoxin(TXN),thioredoxin 2(TXN2),thioredoxin domain-containing protein 5(TXNDC5),thioredoxin domain-containing protein 17(TXNDC17)and hydroxyphenylpyruvate dioxygenase(HPD).We named them as AbNF-κB、HdBCL10、HdIRAK4、HdTAK1、HdMYD88、HdNKIRAS2、HdNKRF、HdTRAF6、HdTRAF2、HdTLR2、HdTLR4、HdTLR6、HdTXN、HdTXN2、HdTXNDC5、HdTXNDC17 and HdHPD.Under the condition of thermal,hypoxia and thermal plus hypoxia stress,the expression levels of these genes were significantly changed.In addition,hierarchical clustering of all these genes under different stresses was analyzed in different organs of H.diversicolor.Moreover,after the inhibit of SaIκB,the expression levels in most of these genes were signifacantly changed than the blank control group and GFP group in culture haemocytes.All these genes were established in a molecular network by Cytoscape under different stresses at 96 h and after the inhibit of SaIκB.