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cDNA Cloning and Expression Analysis of CYP4C and EcR Gene of Portunus Trituberculatus

Author: ZhangXiaoYan
Tutor: LiJian
School: Shanghai Ocean University,
Course: Clinical Veterinary Medicine
Keywords: Portunus trituberculatus Cytochrome CYP4 Ecdysteroidreceptor EcR Salinity stress Embryonic development Gene cloning Expression
CLC: S917.4
Type: Master's thesis
Year: 2013
Downloads: 22
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Abstract


The growth and development of crustaceans were accompanied by periodicecdysis activities, and ecdysis was the symbol of crustaceans. The previous studiesindicated that ecdysis was regulated by molting hormone, and the process of ecdysisinvolved in the complicated physiological process, including many enzymes,hormones and regulatory factors, were regulated by both endocrine systems andnervous systems. Cytochrome CYP4C is an important metabolic enzymes in vivo, andthe study focused on the function and metabolism of it in marine crustaceans, whichinduced by exogenous substances such as polycyclic aromatic hydrocarbon andmarine pollutants, and endogenous substances such as molting hormone. Ecdysteroidreceptor EcR gene is the target of molting hormone, and plays an important role in theregulatory of moulting process. At present, the study on the relationship with ecdysisin CYP4C and EcR gene were few reported in crustaceans, and the research of impactby the rapid changes of environmental factors was also few reported.In the present study, the cytochrome CYP4C and ecdysteroid receptor EcR geneof Portunus trituberculatus were obtained through RT-PCR and SMARTTMRACEtechnology, and the relative expression of CYP4C and EcR were investigated in P.trituberculatus after salinity stress. Beside this, the relative expression of EcR inembryonic development of P. trituberculatus was also studied. The present study willcontribute to the study of environmental suitability and ecdysis mechanism in P.trituberculatus.The complete cDNA sequence of CYP4C gene in P. trituberculatus was firstcloned through RT-PCR and SMARTTMRACE technology. The full-length of theCYP4C gene was1890bp and encoded the protein of514amino acids.Bioinformatics analysis deduced the CYP4C gene protein molecular weight of59.54k Da with a theoretical pI8.08. The amino acid sequence had CYP conserveddomains of helix K (ExxR)and heme-binding motif(FxxGxxxCxG). BLASTanalysis revealed that the similarities of CYP4C with Carcinus maenas, Litopenaeus vannamei, Macrobrachium nipponense、Fenneropenaeus chinensis、Orconecteslimosus and Cherax quadricarinatus were88%、72%、66%、59%、60%and59%,respectively. Real-time fluorescent quantitative PCR was used to assess the mRNAexpression of CYP4C in different tissues and its expression level of CYP4C after thesalinity stress. The results showed that CYP4C expressed in all the tested tissues of P.trituberculatus, including hepatopancreas, muscle, eyestalk, gills and heart. Thehighest expression level was observed in hepatopancreas, while the lowest was inheart. The expression level of CYP4C was up-regulated distinctly in thehepatopancreas of P. trituberculatus at salinity of45and gradually reduced with thetime extended that achieved the level of control group after48h; CYP4C expressiongradually reduced with the time extended at salinity of11and significantly lowerthan those of the control group at the6h. The results implied that CYP4C mightparticipate in the salinity accommodation and play an important role in the moltingmechanism.The complete cDNA sequence of EcR gene in P. trituberculatus was firstcloned through RT-PCR and SMARTTMRACE technology. The full-length of theEcR gene was2996bp and encoded the protein of503amino acids. Bioinformaticsanalysis deduced the EcR gene protein molecular weight of55.69kDa with atheoretical pI7.33. BLAST analysis revealed that the similarities of EcR withCarcinus maenas, Scylla paramamosain, Uca pugilator, Gecarcinus lateralis,Homarus americanus, Crangon crangon and Marsupenaeus japonicas were94%,90%,88%,84%,82%,73%and66%, respectively. Real-time fluorescentquantitative PCR was used to assess the mRNA expression of EcR in differenttissues and its expression level of EcR after the salinity stress. The results showedthat EcR expression existed in all tested tissues of P. trituberculatus, includinghepatopancreas, muscle, eyestalk, gills and heart. The highest expression level wasobserved in hepatopancreas, while the lowest was in heart. The expression level ofEcR was significantly lower than those of the control group at salinity of45after24h and declined generally. At salinity11, the EcR expression presented the trend ofrising after the first decreases and reaches the lowest level at12h. After that theexpression level rose gradually and significantly higher than those in the controlgroup after48h.Real-time fluorescent quantitative PCR was used to assess the mRNA expressionof EcR in embryo of P. trituberculatus, which were in the cleavage stage, blastula stage, gastrula stage, nauplius earlier stage, nauplius later stage and zoea stage,respectively. The results indicated that EcR were expressed in all of the embryonicdevelopment stage. The expression level of EcR dramatic increased from blastulastage to gastrula stage and arrive peak in gastrula stage. After decreased in naupliuslater stage, the expression level of EcR rised again. The present study revealed thatEcR involved in physiological reaction, such as organ formation and differentiation,and might play an important role in the process of embryonic development.

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CLC: > Agricultural Sciences > Aquaculture, fisheries > Aquatic basic science > Aquatic Biology > Aquatic Zoology
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