The Characterization Of Ferritin And HSP70Gene Of Exopalamon Carincauda Be Treated With WSSV And Dry Exposure

Ferritin gene is an important functional gene in regulating growth and immunity,which regulates iron metabolism, cellular antioxidative protective mechanisms, andtumor immunity. So, ferritin research has became a hotspot in biology. Heat shockprotein70(HSP70) is core member in heat shock protein family, and one of the mostimportant stress proteins in Crustacea. Currently, most of the research on ferritin andHSP70are confined in vertebrates, while there is a paucity of study in invertebrates,especially in crustacean. Exopalamon carincauda were used as subjects in this study,ferritin gene were cloned, and the effects of WSSV stimulation on ferritin geneexpression were analyzed. More over, survival rate and expression of ferritin and HSP70genes in E. carincauda under Dry Exposure were also calculated. This study mainlyaims at understanding the immune characters of crustacean and the response of E.carincauda under environmental stress. This study is composed of following two parts.The first part of the study uses E. carincauda as subject; the cDNA sequence offerritin gene (Ecfer) with755bp was obtained with RT-PCR and RACE. Noncodingregions of this gene in5′and3′end are112bp and146bp respectively, with an ORF of497bp. The protein of ferritin gene was speculated to have169amino acid residues, amolecular mass of19.1kDa, an isoelectric point of5.64, and an iron response element(IRE) in5′end of noncoding region. The amino acid residues sequence comparisonrevealed a homology of64%-67%with other crustacean animals, and53%-57%in Hchain of ferritin in vertebrates. A phylogenetic tree created according toNeighbor-joining proves that the amino acid sequence of Ecfer gene merges with crustaceans. Fluorescence quantitative PCR was used to study the distribution of Ecfergene expression and variation in hemolymph, hepatopancreas after WSSV stimulation.Ecfer was located in hepatopancreas, ovary, muscle, gill and hemolymph, among them,hepatopancreas has the most expression, while the ovary has the least. Up regulation(P<0.05) of ferritin in hepatopancreas and hemolymphwas detected after WSSVinjection after3h and6h, indicating WSSV can significantly up-regulate Ecfer geneexpression. The previous results suggest that Ecfer gene may play an important part inthe immune reaction against WSSV in E. carincauda, and that hemolymph is moresensitive to WSSV than hepatopancreas.The second part of this study uses E. carincauda to conduct Dry Exposureexperiment, and survival rates after12h were documented. E. carincauda were dividedinto four experimental groups, including dry and low temperature (group C), dry androom temperature (group CG), humid and low temperature (group P), humid and roomtemperature (group PG), and one control group (group D). RT-PCR was applied tomeasure the ferritin and HSP70gene expression in hemolymph, hepatopancreas andmuscle after different treatments.(1) Group P, which keeps moist and lowerstemperature with ice, has a survival rate of75%, significantly higher than otherexperimental groups (P<0.05).(2) Dry Exposure stress up regulates the expression offerritin gene inhemolymph, hepatopancreas and muscle, notably higher than the controlgroup (P<0.05). The up regulation of ferritin gene comes in a time order, hemolymphappears the first. Dry Exposure stress prominently down regulates the expression offerritin gene in group C, group CG, and group PG, than control group (P<0.05).(3) DryExposure stress up regulates the expression of HSP70gene inhemolymph,hepatopancreas and muscle, notably higher than the control group (P<0.05). The upregulation of HSP70gene comes in a time order, hepatopancreas appears the first. Themost significant up regulation of HSP70gene in group C, group CG, and group PG,appears in1.5h after treatment, much higher than group P (P<0.05). previous resultsindicates that humid and low temperature could increase the survival rate of Dry Exposure after12h, which can directly effect the expression of ferritin and Hsp70genes.Ferritin and Hsp70could act as antioxidant, and be used as physiological indices of antiDry Exposure stress in E. carincauda.