Shuhong Wang

Molecular cloning and expression of interleukin-1 receptor-associated kinase 4, an important mediator of Toll-like receptor signal pathway, from small abalone Haliotis diversicolor

Mammal interleukin-1 receptor-associated kinases (IRAKs) have been demonstrated to play important functions in TLRs (Toll-like receptor) signal pathway and T cell proliferation, but there is less knowledge available on mollusc IRAKs. In this study, a molluscan IRAK-4 gene, saIRAK-4, was cloned for the first time from the small abalone (Haliotis diversicolor). Its full-length cDNA sequence was 2062 bp, with a 1548 bp open reading frame encoding a protein of 516 aa. The molecular mass of the deduced protein was approximately 57.8 kDa with an estimated pI of 5.23, and showed highest identity (47%) to acorn worm Saccoglossus kowalevskii. Amino acid sequence analysis revealed saIRAK-4 shares conserved signature motifs with other IRAK-4 proteins, including the death domain (DD), serine/threonine/tyrosine protein kinase domain (STYKc), protein kinases ATP-binding region signature, serine/threonine protein kinases active-site signature and prokaryotic membrane lipoprotein lipid attachment site. Quantitative real-time PCR was employed to investigate the tissue distribution of saIRAK-4 mRNA, and its expression in abalone under bacteria challenge and larvae at different developmental stages. The saIRAK-4 mRNA could be detected in all examined tissues, with the highest expression level in gills, and was up-regulated in hemocytes and gills after bacteria injection. Additionally, saIRAK-4 was constitutively expressed at all examined developmental stages. These results indicate that saIRAK-4 could respond to pathogenic infection and may play an important role in the adult abalone immune system and early innate immunity in the process of abalone larval development.

Expressed sequence tag analysis for identification and characterization of genes related to Tributyltin (TBT) exposure in the abalone Haliotis diversicolor supertexta.

The analysis of expressed sequence tags (ESTs) is an efficient approach for gene discovery, expression profiling, and the development of resources for functional genomics. To analyze the transcriptome response to tributyltin of small abalone Haliotis diversicolor supertexta, a normalized cDNA library of hepatopancreas after exposure of animals to tributyltin was constructed. Three thousand and forty eight high quality ESTs were generated. After processing, a total of 2473 unigenes comprising 370 contigs and 2103 singlets were acquired. BLAST identified 1108 clones (45%) as known genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Simple Sequence Repeat (SSR) identification were also carried out to acquire more information on pathway and EST-SSR. According to GO categories, the total ESTs distribute across the various functional categories. In addition, 43 potential toxicology-related clusters were identified. This work provides the first set of genetic data for small abalone which is of great value for further exploitation of this species in functional genomics and toxicogenomics. Our interesting results will be mainly useful to choose biomarkers for ecotoxicological studies.

Characterization of interleukin-1 receptor-associated kinase 1 binding protein 1 gene in small abalone Haliotis diversicolor.

Interleukin receptor-associated kinase (IRAK)-1 binding protein 1 (IRAK1BP1) is a critical factor in preventing dangerous overproduction of proinflammatory cytokines by the innate immune system and in influencing the specificity of TLR responses. In this study, a first molluscan IRAK1BP1 gene, saIRAK1BP1, was cloned from the small abalone (Haliotis diversicolor). Its full-length cDNA sequence is 1047bp, with a 747bp open reading frame encoding a protein of 249 aa. The molecular mass of the deduced protein is approximately 28.1kDa with an estimated pI of 8.87, and shows highest identity (52%) to acorn worm Saccoglossus kowalevskii. Amino acid sequence analysis revealed that saIRAK1BP1 shares a conserved SIMPL domain. Quantitative real-time PCR was employed to investigate the tissue distribution of saIRAK1BP1 mRNA, and its expression in abalone under bacteria challenge and larvae at different developmental stages. The saIRAK1BP1 mRNA could be detected in all examined tissues, with the highest expression level in hemocytes, and was up-regulated in gills, kidneys and hemocytes after bacteria injection. Additionally, saIRAK1BP1 was constitutively expressed at all examined developmental stages. These results indicate that saIRAK1BP1 play an important role in the adult abalone immune system and might be essential in embryo and larval development in abalone.

Identification and expression analysis of immune-related genes linked to Rel/NF-κB signaling pathway under stresses and bacterial challenge from the small abalone Haliotis diversicolor

Inhibitor of NF-κB (IκB), nuclear factor-κB (NF-κB), and Akirin2 are all important members of Rel/NF-κB signaling pathway, which plays a pivotal role in regulating the innate immune response of vertebrates and invertebrates. In this study, the IκB (SaIκB) and Akirin2 (SaAkirin2) cDNAs of small abalone Haliotis diversicolor were cloned and characterized. The full length cDNA of SaIκB and SaAkirin2 were 1748 bp and 1452 bp respectively, encoding a protein of 401 aa and 187 aa respectively. A conserved degradation motif (DS56GIYS60) and six ankyrin repeats were identified in the SaIκB by SMART analysis. Meanwhile, a typical nuclear localization signal (NLS) was found at the N-terminal region of the SaAkirin2 protein. Also, the mRNA expression level of SaIκB, SaAkirin2, and AbNF-κB were detected by quantitative real-time PCR. The results revealed that all these three genes were ubiquitously expressed in 7 selected tissues. The expression level of SaIκB in gills was higher than that in other tissues (P < 0.05) while the expression level of AbNF-κB was significantly higher in hepatopancreas and haemocytes. The highest expression level of SaAkirin2 was detected in hepatopancreas, followed by mantle. The mRNA expression levels in either gills or haemocytes of SaIκB, SaAkirin2, and AbNF-κB were significantly up-regulated (P < 0.05) post thermal stress, hypoxia exposure, thermal plus hypoxia stress and the injection of Vibrio parahaemolyticus. These results indicated that these three NF-κB signaling pathway-related genes are involved in response to bacterial infection and play essential roles in response to thermal and hypoxia stress.

Molecular cloning and expression of allograft inflammatory factor 1 in Haliotis diversicolor under stresses

Allograft inflammatory factor-1(AIF-1)is a kind of calcium-binding protein with EF-hand domain.AIF-1 mRNA is robustly induced by IFN-γ in murine macrophages.It is involved in transplant rejection,immune inflammatory reaction,non-inflammatory injury and so on.In this study,a molluscan AIF-1 gene,HdAIF-1,was cloned for the first time from Haliotis diversicolor.Its full-length cDNA sequence is 942 bp,with a 456 bp open reading frame encoding a protein of 151 aa.Quantitative real-time PCR results indicated that HdAIF-1 could be detected in all examined tissues,with the highest level in hemolymph and gill.Under thermal stress,HdAIF-1 was up-regulated in gill significantly at temperature-rise period and achieved the highest level when up to 31 ℃.However,the expression level of HdAIF-1 in hemolymph and hepatopancreas did not show significant difference between thermal group and control group from the first phase to the fourth,and its expression level was up-regulated significantly at 96 h in these two tissues.Under hypoxia stress,the expression level of HdAIF-1 in hemolymph showed no significant difference between control and exposed groups.However,it was down-regulated at 24 h and up-regulated at 192 h remarkably in gill.After Vibrio parahaemolyticus challenge,the HdAIF-1 expression level achieved the highest level at 3 h in hemolymph,reduced to the control level at 12 h,and then up-regulated significantly at both 24 h and 48 h.These results suggest that HdAIF-1 may play important roles as an immune factor under different stresses.

Development of Phase Separation Immunoassay

Abstract Phase separation immunoassay (PSI) combines some advantages of both homogeneous and heterogeneous immunoassays by means of a fast homogeneous immune reaction and a simple heterogeneous separation process. The method is readily amenable to automation. The principle of PSI is introduced. The carriers, the immobilization modes, the labels, the applications, and the prospect of PSI are comprehensively reviewed on the basis of 60 references cited.

Identification and expression of transcription factor sox2 in large yellow croaker Larimichthys crocea

As an important transcription and pluripotency factor, Sox2 plays its functions essentially in the regulation of self-renewal and pluripotency of embryonic and neural stem cells, as well as embryogenesis, organogenesis, neurogenesis and regeneration. The data is lacking on Sox2 in large yellow croaker (Larimichthys crocea) (Lc-Sox2) which is a limitation on the generation of induced pluripotent stem cells (iPSCs). In this study, Lc-sox2 was cloned by RACE (rapid amplification of cDNA ends) and analyzed by Bioinformatics. The quantitative real-time PCR (qRT-PCR) and whole mount in situ hybridization (WISH) were used to detect the expression of Lc-sox2. The full-length cDNA sequence of Lc-sox2 is 2135 bp and encodes a 322-aa (amino acids). Lc-Sox2 possesses a highly conserved HMG-box as DNA-binding domain, maintains highly conserved with vertebrates, particularly with teleosts. In tissues, Lc-sox2 was expressed with gender difference in brain and eye (female > male), in embryos, Lc-sox2 was expressed with a zygotic type that the high level expression began to appear in the gastrula stage. The spatio-temporal expression patterns of Lc-sox2 suggested the potential involvement in embryogenesis, neurogenesis, gametogenesis and adult physiological processes of large yellow croaker. Our results contributed to better understanding of Sox2 from large yellow croaker.

miR-34 regulates reproduction by inhibiting the expression of MIH, CHH, EcR and FAMeT genes in mud crab Scylla paramamosain : ZHOU et al.

Mud crab Scylla paramamosain is a commercially important species widely cultured in China. It is well known that the eyestalk regulates reproductive activities in crustaceans. In our previous research, we found that the miR‐34 expression level in male eyestalk was significantly higher than that in females. Thus, we assumed that it may play an important role in regulating reproduction. In this study, we used bioinformatic tools to identify the target genes of miR‐34 in eyestalk. Six reproduction‐related genes with an intact 3′‐untranslated region (UTR), including molt‐inhibiting hormone (MIH), crustacean hyperglycemic hormone (CHH), vitellogenesis‐inhibiting hormone, red pigment concentrating hormone, ecdysone receptor (EcR), and farnesoic acid methyltransferase (FAMeT) were identified. When the 3′‐UTR plasmid vectors of the six genes were cotransfected with miR‐34 mimics into 293FT cells, respectively, the luciferase activities of four genes (MIH, CHH, EcR, and FAMeT) were significantly decreased compared with that in the control group; on the contrary, when the six plasmid vectors were cotransfected with the miR‐34 inhibitor respectively, the luciferase activities of four genes (MIH, CHH, EcR, and FAMeT) were significantly higher than that in the control group. When agomiR‐34 and antagomiR‐34 were injected into the eyestalk respectively in vivo, the expression levels of the MIH, CHH, EcR, and FAMeT genes were detected by a quantitative real‐time polymerase chain reaction. The results showed that agomiR‐34 suppressed the expression of the four genes, whereas antagomiR‐34 enhanced their expression. These experimental results confirmed our hypothesis that miR‐34 may indirectly regulate reproduction via binding to the 3′‐UTRs of MIH, CHH, EcR, and FAMeT genes and suppressing their expression.