Construction, Analysis Of The Transcriptome Library, And Cloning, Expression, Antibacterial Activity Study Of Immune-related Genes In Scapharca Broughtonii

The ark shell Scapharca broughtonii(Mollusca, Bivalve, Arcoida), one of the most important marine commerical bivalve species, mainly distributes in the coasts of Bo Hai Sea and North of Yellow Sea, China. Because of its large body, rapid growth rate, delicious tastes and high protein and vitamin contents, the export in the exchange rates of ark shell is higher in the aquatic products, and S.broughtonii has become one of the most popular farming mollusks in North China due to its high economic value in recent years. The wild resources were rich in the early years, and the individuals in the markets were fished. However, with the deterioration of marine ecological environment resulted from the extended farming scale and frequency human activities in coastal waters, and over-fishing, the wild resources decreased, and the disease caused by various pathogens occured. The artificial propagation and artificial releasing of ark shells were ways to recover the resources. Therefore, the better understanding of the innate immune abilities and immune defense mechanisms of ark shell will be beneficial to the development of health management and disease control in the ark shell aquaculture. However, the researches about innate immune were just the classification and immune function of hemocytes, and other aspects were blank in the domestic and overseas.In the present study, a transcriptome library was constructed by Illumina HiseqTM 2000 technology and analyzed the bioinformation. The de-novo assembly was carried out by Trinity software and 86,689 high quality unigenes(>200 bp) with total 71,873,603 bp nucleotides were obtained, the mean length was 829 bp, the N50 length was 2256 bp, the N90 was 454 bp, and the longest unigene was 33,269 bp. The results of genes annotation showed that 22,117(25.51%) unigenes had similar sequences in the Nr, and 21,404(24.69%), 21,207(24.46%) unigenes were annotated successfully in the GO and PFAM, respectively. The massive unigenes of ark shell provided important information of the immune system and laid the groundwork for development of molecular markers related to disease resistance in bivalve species.In this study, a full-length Mn SOD c DNA of S.broughtonii was cloned by RACE method(termed Sb Mn SOD). The c DNA contained a 32 bp length 5’-UTR, a 275 bp length 3’-UTR and an open reading frame(ORF) of 696 bp which encoded a polypeptide of 232 amino acids. The predicted molecular weight(Mw) was 25.67 k Da, and the theoretical isoelectric point(p I) was 7.13. Four putative amino acid residues(His-57, His-105, Asp-190 and His-194) responsible for manganese coordination were located in the most highly conserved regions of Sb Mn SOD and the signature sequence(DVWEHAYY) also existed in Sb Mn SOD. The deduced amino acid sequence of Sb Mn SOD shared high identity to Mn SOD from other species. The m RNA expression profiles of Sb Mn SOD in tissues of foot, gill, mantle, adductor muscle, hemocytes and hepatopancreas analyzed by quantitative Real-time PCR(q RT-PCR) suggested the transcripts of Sb Mn SOD distributed in foot, mantle, gill, hemocytes, adductor muscle, and hepatopancreas. Importantly, Vibrio anguillarum challenge resulted in the upregulated expression of Sb Mn SOD m RNA, and showed obvious time dependence, indicating Sb Mn SOD actively participated in the immune response process. What’s more, further analysis on the antibacterial activity of the recombinant Sb Mn SOD showed that the fusion protein could remarkably inhibit growth of both Gram-positive and Gram-negative bacteria.A full-length ferritin of S.broughtonii was cloned by RACE method(termed Sb Fer): the c DNA contained a 5’- UTR of 182 bp, a 3’-UTR of 229 bp and an open reading frame of 519 bp which encoded a polypeptide of 172 amino acids. The predicted molecular weight was 20.0 k Da, and the theoretical isoelectric point(p I) was 5.16. Three introns with length of 902 bp, 373 bp, 402 bp embedded in four exons. Iron response element(IRE) existed in the 5’-UTR. The deduced amino acid sequence of Sb Fer possessed almost all characteristic sequences of vertebrates H type ferritin, shared 63%- 96% identity to other analyzed mollusks, and shared higher identity with H type ferritin of vertebrates than L type ferritin. q RT-PCR results showed the ferritin m RNA expressed in all examined tissues of the ark shell in the order of hemocytes, foot, mantle, gill, adductor muscle, and hepatopancreas. Interestingly, V.anguillarum challenge resulted in significant up-regulation of Sb Fer m RNA, except in foot, and showed an obvious time dependence. And antibacterial activity test of the fusion protein indicated that recombinant Sb Fer could function as an immune antibacterial agent to both Gram-positive and Gram-negative bacteria.The HSP90 c DNA of S.broughtonii was cloned(termed Sb HSP90) by RACE method. The c DNA contained a 5’- UTR of 195 bp, a 3’-UTR of 325 bp and an open reading frame of 2187 bp which encoded a polypeptide of 728 amino acids. The predicted molecular weight was 83.72 k Da, and the theoretical isoelectric point was 4.85. The amino acids sequence processed five signature sequence of HSP90 family, ATPase domain and highly conserved MEEDV in the C-terminal. The deduced amino acid sequence of Sb HSP90 shared high identities to HSP90 from other mollusks was over 83%, and the identities with HSP90-α from vertebrates were very similar to HSP90-β. q RT-PCR results showed that the Sb HSP90 gene expressed in all examined tissues, and the highest expression level was observed in foot. After being challenged by V. anguillarum, the expression level of Sb HSP90 was up-regulated significantly, and showed an obvious time dependence and instantaneous expression trends.The c DNA of galectin with multiple carbohydrate recognition domains(CRDs) was cloned from S.broughtonii(termed Sb Gal) by RACE method. The c DNA contained a 5’-UTR of 213 bp, a 3’-UTR of 1342 bp and an open reading frame of 1565 bp which encoded a polypeptide of 554 amino acids containing four carbohydrate-recognition domains, which was another specific galectin in bivalves. The predicted molecular weight was 63.32 k Da, and the theoretical isoelectric point was 4.99. The results of multiple sequences alignment and phylogeny analysis showed that the identities of Sb Gal amino acids sequence shared with other galectin from other molluscs was 46%- 70%. And the four domains of Sb Gal shared 36%- 41% identities each other. q RT-PCR showed the m RNA distributed in all tested tissues and the highest expression level was observed in hemocytes. After being challenged by V.anguillarum, the m RNA expression level of Sb Gal was up-regulated significantly, and showed obvious time dependence and instantaneous expression trends.