Cloning And Expression Profile Of Two Immune-related Genes And Studies On The Applicability Of Eleven Internal Reference Genes In Mud Crab, Scylla Paramamosain

Scylla paramamosain, one of the most precious crabs cultured in China, is proneto infection of various microbes which threats the mud crab industry. In this study, wecloned two immunization-associated genes from S. paramamosain, and detected theexpression profiles using Quantitative real-time PCR. The stability of several internalreference genes was also analyzed. The main results are as follows:1. Molecular cloning and expression of mud crab SpMIFAs one of the first found cytokines, macrophage migration inhibitory factor (MIF)plays important roles in several physiological processes in crabs, and has attractedextensive attention recently. In this study, a MIF cDNA (GenBank accession number:JX131610) from mud crab Scylla paramamosain was cloned based on a sequence of S.paramamosain cDNA library. The full length is734bp, consists of a363bp openreading frame, which encodes the SpMIF, a120amino acids peptide chain. SpMIF is13.46kDa in molecular weight, with the pI of6.82. The alignment analysis showedthat SpMIF appeared to be closely related to the counterpart from Eriocheir sinensis(68%). Quantitative real-time PCR analyses revealed that SpMIF was highly expressedin hepatopancreas and hemocytes. Furthermore, after challenged by Vibrioparahaemolyticus (4.00×106CFU/mL), the expression level was increasedsignificantly after6h and peaked at8h, then fell to the common level in48h. Thesedata indicated the successful cloning of SpMIF and suggested its participation in theimmune system of mud crabs.2. Molecular cloning and expression of mud crab SpCLRThe full-length sequence of C-type lectin receptor was cloned from mud crab andsubjected to further investigation. The full-length of SpCLR consists of697bp, with anORF of510bp encoding a peptide of169amino acids. The predicted molecular weight of the putative protein is19.236kDa, and the theoretical isoelectric point is4.57.GenBank accession number: KC902764. Expression level of SpCLR was detectedusing real-time PCR and showed ubiquitous expression in all six organs with thehighest in hepatopancreas. After been treated by V. parahaemolyticus, the temporalexpreesion of SpCLR mRNA were up-regulated from4h, and reached the maximum at12h.3. Studies on the stability of eleven candidate internal reference genesQuantitative real-time transcription-polymerase chain reaction (RT-qPCR) is nowused widely in studies about expression levels. The selection of one or more stablereference gene(s) used for data normalization is crucial. In this study,1) expressionlevels of eleven candidate reference genes (β-actin,16S r RNA,18S rRNA,28S rRNA,α-I tubulin, GAPDH, ribosomal protein L13, elongation factor1α, elongation factor2,arinine kinase and ubiquitin) were detected in six different tissues (hepatopancreas,hemocytes, heart, gill, muscle and testis) using the GenomeLab GeXP analysis system(Beckman Coulter). Gene expression data were analyzed using two different statisticalmodels: geNorm and NormFinder.18S rRNA and elongation factor1α were identifiedas the best two reference genes.2) Expression levels of these genes were detected inthe hemocytes after being challenged by V. parahaemolyticus, and suggested thatubiquitin was the most stable gene after the treatment.18S rRNA, elongation factor1αand ubiquitin were recommended as the best combination.In conclusion, two immune-related genes from mud crab, S. paramamosain werecloned and the expression profiles in different tissues and after immune stimulationwere analyzed by Real-time PCR. The stability of eleven candidate reference geneswas compared to provide a foundation for the more accurate use of RT-qPCR in theanalysis of gene expression.