Gene Cloning,Expression And Preliminary Functional Study Of Class B Scavenger Receptors And Lectins In The Sea Urchin Strongylocentrotus Nudus

Strongylocentrotus nudus,also named Dalian purple sea urchin,belonging to Echinodermata-Echinoidea-Strongylocentrotidae,is an economically important sea urchin in North China Sea,and it is also an excellent native speices.And sea urchins are popular with consumers for its wealth of nutritional and medicinal value.However,up to now,sea urchin aquaculture is still constrained by diseases,germplasm and nutrition.Due to lack of acquired immunity,sea urchins,like other echinoderms,defense against foreign pathogens using innate immune system.Development-related genes cloning and expression level detection will help to promote the healthy culture of sea urchins.Thus,investigation of the genes related to immunity and development could provide necessary theoretical basis for the sea urchin breeding.Class B scavenger receptors and lectins are genes related to both immunity and development.To study these genes in the sea urchin,experiments of molecular cloning,prokaryotic expression,real time PCR and bacterial inhibition were performed.The mains results are shown below:1)Three B-type scavenger receptors and 2 lectins were cloned.Three newly discovered scavenger receptors in the sea urchin were named SRB-1,SRB-2 and SRB-3,respectively.SRB-1 has an open reading frame of 1632 bp encoding 543 amino acids with predicted molecular weight is 61.9kDa,and 3' untranslated region of 315 bp.The ORF of SRB-2 is 1653 bp in lenth,encoding 550 amino acids with predicted molecular weight of 62.9kDa,and its 3'UTR is 220 bp.SRB-3 encodes 503 amino acids from 1512 bp ORF.No signal peptide was found in 3 scavenger receptors.Moreover,analysis was performed on the relationship of sea urchin known microRNAs and scavenger receptors whose 3'UTR were obtained.Combined with miRNA sequencing results and predicted binding sites,SRB-1 and SRB-2 were believed not to be the target genes of any known sea urchin miRNA.2)In two newly identified lectins,galectin-8(Gal-8)has an open reading frame of 939 bp,encoding 321 amino acids with molecular weight 34.98 kDa,isoelectric point of 9.40,while endoplasmic reticulum lectin(E-lectin)contains 1371 bp ORF that encodes 456 amino acids.3)The extracellular regions of SRB-1,SRB-2 proteins,as well as Gal-8,were expressed,.The immune functions of these recombinant proteins were preliminarily investigated.In vitro experiments indicated that recombinant proteins could inhibit the growth of Escherichia coli and Vibrio anguillarum while Vibrio alginolyticus could not be restrained.Real-time PCR technology was performed to identify the expression level of these 3 genes in individuals of different development stage.5 immune-related and development-related genes were cloned,including 3 B-type scavenger receptors and 2 lectins.And recombinant proteins of SRB-1,SRB-2 and Gal-8 were expressed.Our study provides better knowledge of sea urchin innate immunity and development,which will be beneficial for its breeding.