Cloning And Characterization Of SpCI-MPR Gene In Mud Crab

Cation-independent mannose-6-phosphate receptor(CI-MPR) is a multi-functional receptor which subjects to the p-type lectin family. It participates in lots of biological processes, ranging from growth, placental development, tumour suppression and signalling that are essential for normal cellular function. However, previous studies on CI-MPR gene have been mainly confined to vertebrates.Mud crab(Scylla paramamosain) representing one of the crustacean species has a wide geographical and environmental distribution in southeastern coast of China and other areas in Asian. Owing to its great value both in commerce and nutrition, S. paramamosain has been intensively studied over the past years. To date, remarkable achievements have been gained on genetic research. The well-studied background makes mud crab a favorable model for the study of CI-MPR gene in crustacean. The main results in this study were indicated as follows: I. Obtained the complete sequence of Sp CI-MPR geneIn the present study, the CI-MPR gene c DNA sequence of S. paramamosain(designated as Sp CI-MPR thereafter) has been obtained by RACE method. Data has been deposited in the Gen Bank(Accession number: KM658157). The full length of Sp CI-MPR c DNA was 3931 bp encoding a protein of 1095 amino acids and that putative Sp CI-MPR protein had 7 highly repetitive sequences that were referred to as “MRH” domains. Though strikingly different from its vertebrate homologs in overall-length, this protein shared high similarity with its vertebrate homologies on the domain level that each domain has 6 or 8 conserved cysteines which is exclusive for MRH family protein. The BLASTP search showed that CI-MPR shared the highest identity(53%) with partial Marsupenaeus japonicus CI-MPR homologs(AFJ59952.1) on the amino acid level. Meanwhile, the phylogenetic analysis based on the amid acid sequence of CI-MPR protein was consistent with the evolutionary relationship. Based on the above findings, it showed that we have successfully cloned the Sp CI-MPR gene c DNA sequence. II. Characterization the sequence with the method of bioinformaticsWe characterized its structural features and found that this protein had many N-glycosylation sites and phosphorylation sites which would be responsible for Man-6-P binding. The domain 5 and domain 6 of Sp CI-MPR protein had the highest with the domain 3 and domain 9 of the vertebrates’ which play a vital role in Man-6-P binding. The domain 5 and domain 6 might be therefore play import roles in the binding of Man-6-P in S. paramamosain. Meanwhile, we speculate that Sp CI-MPR might not be capable of recognizing IGF-II since this protein lacks hydrophobic pocket and some other crucial binding sites for IGF-II binding. III. Investigated the spatial and tissue distributionTo shed light on the role of Sp CI-MPR gene in the early postnatal development and growth of mud crab, we quantified its expression level from the stages of zoea-I to megalopa, since this window was more likely to reflect the growth and development potential of Sp CI-MPR gene measure functionally relevant expression levels. Our results revealed that Sp CI-MPR gene was developmental stage specifically expressed which reached a peak by the stage of zoea-Ⅱ whereas rapidly declined after that. Besides, tissue distribution was analyzed in eleven typical tissues, namely hepatopancreas, blood, heart, ovary, testis, gill, muscle, stomach, connective tissue, cerebral ganglia and thoracic ganglia. We found that Sp CI-MPR gene was highly expressed in hapatopancreas, blood and heart; while with lower transcript level in the other examined tissues(p=0.00002). Taken these findings altogether, it suggests that Sp CI-MPR is an imprinting gene crucial for the early development and growth in mud crab. IV. Investigate tissue and allelic expression pattern of Sp CI-MPR gene and itsrelationship with the expression levelAt the same time, we found that Sp CI-MPR gene contained a Cp G island in 5’ portion which might be the potential methylated region. We examined the expression of the alleles of this gene. Given the complex imprinting of CI-MPR presented in the known organisms therefore we explored the imprinting status of Sp CI-MPR gene on tissue level to determine its exact imprinting pattern in mud crab. Meanwhile, we examined its imprinting status and transcript level in different tissue. Our results suggested that Sp CI-MPR was an imprinted gene acting in a tissue specific manner, and that its transcript level in each tissue was largely associated with its imprinting status. This result suggests that imprinting might severe as a modulator to regulate the transcript level of Sp CI-MPR gene. V. Generated the fusion Sp CI-MPR protein by constructed a recombinantplasmidFurthermore, we constructed a recombinant plasmid and successfully generated the fusion Sp CI-MPR protein in competent Escherichia coli BL21(DE3) cell which will facilitate the further study of Sp CI-MPR on protein level.