Investigation Of The Role And Mechanism Of Ferritin In Enhancing Stress Resistance In A New Variety Of Yesso Scallop (Patinopecten Yessoensis),"Haida Golden Scallop"

1Proteomic analysis identifies proteins related to stress resistance in Yessoscallop (Patinopecten yessoensis)Compared with outside environment, pathogens, protozoan, and predators, geneticstructure is a most fundamental factor to influence the survival rate of Yesso scallop.In order to keep sustainable development of breeding, it is urgent to improve andbreed a new variety with higher resistance. Most recently, based on molecular markers,like resistant genes, breeding has been promoted and got better results. A new variety,“Haida golden scallop”, with higher content of carotenoids and higher resistancecompared to the common Yesso scallop, has been developed successfully by ourgroup. To unravel the molecular mechanism of high stress resistance in “Haida goldenscallop”, a proteomic approach was applied in this study to explore the differencesbetween the new variety and common individuals. It resulted in7differentiallyexpressed proteins. Real-time PCR showed that four of the corresponding genes werealso significantly up-regulated at the mRNA level in the new variety. These resultsindicate that:1) Genes involved in various biological processes, such as lipid andglucose metabolism, protein-folding and degradation, were altered.2) Carotenoidsaccumulation may influence peroxisome proliferator-activated receptor (PPAR)signaling pathway, and PPARs may play a vital role in these above changes.3)Ferritin, as an important factor in innate immune is a good candidate for unravelingthe mechanism of high stress resistance in “Haida golden scallop”. This studyrepresents a starting point for future work concerning the genetic basis of carotenoid accumulation across all marine mollusks.2Identification and characterization of six ferritin subunits involved in immunedefense of the Yesso scallop (Patinopecten yessoensis)As a primary iron storage protein, ferritin plays a vital role in iron homeostasis andinnate immunity. In this study, the DNA and full-length cDNA sequences of sixferritin subunits (PyFer1, PyFer2, PyFer3, PyFer4, PyFer5, and PyFer6) were clonedfrom the Yesso scallop, Patinopecten yessoensis. PyFer1, PyFer2, PyFer3, andPyFer4consist of four exons and three introns；PyFer5is composed of seven exonsand six introns, and PyFer6has a five-exon/four-intron structure. Minisatellite DNAsare found in intron2of PyFer4and PyFer6. Except for PyFer5and PyFer6, the restfour ferritins (PyFer1, PyFer2, PyFer3, and PyFer4) contain putative iron-responseelements (IREs) in their5’ UTRs. Meanwhile, multiple A+U-destabilizing elements(TATT or ATTTA) are present in the3’ UTRs of PyFer2, PyFer4and PyFer6. Theopen reading frames (ORFs) of the six ferritins are522,516,516,687, and663bp inlength, encoding173,171,171,172,228, and220amino acids, respectively. Theseproteins have typical ferritin structures, with four long α-helices, one short α-helix andan L-loop. Four of the predicted proteins (PyFer1, PyFer2, PyFer3, and PyFer4)possess both the ferroxidase center of mammalian H ferritins (E Y E E H E Q) and theiron nucleation site of mammalian L ferritins (D E E), and the four recombinantproteins (rPyFer1, rPyFer2, rPyFer3, and rPyFer4) possess apparent ferroxidaseactivity. Moreover, PyFer5only contains the ferroxidase center, and PyFer6has anincomplete ferroxidase center. PyFer1, PyFer2, PyFer3, and PyFer4are likely tobelong to cytoplasmic ferritins, while both PyFer5and PyFer6possess signal peptides,suggesting that they are secreted ferritins, which is in accordance with the result ofphylogenetic analysis. Quantitative real-time PCR analysis revealed that theexpression of the six PyFers was significantly elevated at the D-shaped stage and wasrelatively high in the adult mantle and hepatopancreas. Furthermore, the six PyFerswere significantly up-regulated by iron or bacterial challenge, and all four purifiedrecombinant PyFers (rPyFer1, rPyFer2, rPyFer3, and rPyFer4) were able to inhibit the growth of the scallop pathogen Vibrio anguillarum. Immunohistochemical analysisshowed that ferritins were widely distributed in various cell types of Yesso scallop.These results suggest that these PyFers are likely to play important roles in manyfundamental biological processes in P. yessoensis, including immune defense, ironhomeostasis, and shell development.3Role of ferritin in the high sresistance of “Haida golden scallop”As primary iron storage proteins, the six ferritins have been demonstrated to playvital roles in the immune defense, iron homeostasis, and shell development of P.yessoensis. In this study, relative mRNA expressions of the six PyFers were evaluatedby iron or bacterial challenge between “Haida golden scallop” and the commonindividuals. Results showed that all six ferritin subunits from “Haida golden scallop”were more sensitive to the ferrous iron or V. anguillarum than those from the commonindividuals, indicating that ferritins might participate in the high resistance of “Haidagolden scallop”.