Study On The Immune Function And Physiological Indication Function With Implantation Of Two Galectins From Hyriopsis Cumingii

The bivalve mollusk Hyriopsis cumingii is the most economically important freshwater pearl-producing mussel in China.To culture pearls,allografts of mantle tissue from a donor mussel are transplanted into the body of a host mussel in a process called implantation.After a series of immune responses,a thin layer of epithelial cells forms around the mantle,creating a pearl sac.Over time,a pearl is produced inside the pearl sac.However,implantation can inflict extensive damage to the mussel and can even cause the animal's death.In addition,infections by pathogenic microorganisms can kill these mussels.How to reduce the risk of infection after the implantation,the identification of allogeneic,pearl formation has become an important topic in pearl culture.The change of immune gene expression can reflect the immune physiological process,and has the function of indication.Cloning and identification of genes associated with the formation of pearl by molecular biological methods can provide new ideas for practical production.Lectins can not only be used as pattern recognition receptors to identify and eliminate pathogen but also involved in the formation of pearls.In this study,we identified two galectins from Hyriopsis cumingii and studied their structure and function comprehensively and systematically.The main results as followed:(1)Cloning and structural analysis of the full-length cDNA sequence of two galectinsWe named two genes named HcGal1 and HcGal2 respectively.The full-length cDNA of the HcGal1 sequence was 1917 bp,including a 140 bp 5'-UTR(untranslated region),a 939 bp ORF encoding 312 amino acids,and an 838-bp 30-UTR that includes a polyadenylation signal sequence(AATAAA)and a poly-A tail.The HcGal1 amino acid sequence contained two CRDs.The HcGal1 molecular weight and theoretical isoelectric point were predicted to be 35.5 kDa and 9.54 respectively,and no signal peptide was identified.The amplicon for HcGal2 consisted of a 797 bp fragment that included an 80 bp 5'-UTR,267 bp 3'-UTR,and an open reading frame of 450 bp.This was predicted to encode a protein of 149 amino acids with a molecular mass of approximately 17.2 kDa and a theoretical isoelectric point of 7.82.The HcGAL2 amino acid sequence contained one CRD.(2)Tissue-specific expression of two galectinsBy qRT-PCR,HcGal1 and HcGal2 transcript levels were detected in all tissues examined.The HcGal1 transcripts were most abundant in the hemocytes.The HcGal2 transcripts were most abundant in the mantle.Interestingly,there was a discrepancy between the mRNA expression results by qRT-PCR and protein enrichment observed by Western blotting.We suggest that this discrepancy between the mRNA and protein expression domains might be due to post-transcriptional control.This post-transcriptional regulation could be due to changes in translational efficiency and/or stability of the mRNA.(3)Genes expression following LPS and bacterial challengesExposure to LPS,A.hydrophila,P.fluorescens and S.aureus all induced high levels of HcGal1 and HcGal2 expression,strongly suggesting an immune-related role for this gene in H.cumingii.The results showed that galectin was a pattern-recognition receptor that participated in the innate immune response against pathogenic microbes.(4)Study on the protein functional of two galectinsThe rHcGal1 and rHcGal2 protein was recombinantly expressed and purified from the E.coli strain BL21(DE3),microorganism agglutination activity assay showed that two recombinant proteins were able to agglutinate some bacteria,suggesting that they are able to identify and resist the invasion of pathogenic microorganisms.The recombinant proteins was injected into the mussels to detect the phagocytosis of hemocytes to "non-self",results showed that the injection of recombinant protein in phagocytosis efficiency than the control group significantly increased,indicating that the recombinant protein of two galectins can promote phagocytosis of hemocytes.(5)Temporal expression pattern of HcGal1 and HcGal2 after implantationAfter implantation,two galectins showed different expression patterns in different tissues,suggesting that HcGal2 gene has a strong immune rejection response to "non-self",and HcGal1 gene may be involved in the negative feedback regulation of the body,which can reduce the level of mRNA and save the donor cells.In addition,following implantation,the host mussels underwent three physiological stages: acute-stress stage(0-24 h),the stage for wound healing and pearl-sac formation(24 h-7 d),and the stage for pearl secretion(7-35 d).The change of immune gene expression can reflect the immune physiological process,and has the function of indication.To study the relationship between expression of immune genes and physiological response of the body after implantation,and then to adjust the breeding environment in time to reduce the rate of infection and failure of implantation for improving the rate of survival and pearl quality.