Identification And Expression Analysis Of Cyprinus Carpio L. Romo1

Romo1is localized in the mitochondria and increases cellular ROS levels via mitochondrialROS production through complex III of the mitochondrial electron transport chain. In thenormal conditions, Romo1is responsible for the balance of ROS levels to maintain redoxhomeostasis, promote the aging cells and apoptosis of cancer cells. What’s more,Romo1-derived ROS levels are involved in the cell proliferation through the regulation of P27expression. Therefore, Romo1is beneficial to the cell growth and survival. In the abnormalconditions, such as serum free state and the stimulation of anti-cancer drug, Romo1will increasecellular ROS levels which induce oxidative damage, tumor proliferation, resulting in a variety ofpathological disorders.ROS have an important function in cell death and cell survival. Most ROS are produced in thecomplex I and III of the mitochondrial electron transport chain. The physiological range ofreactive oxygen species (ROS) concentrations is intracellularly maintained from a low of~0.001μΜ to a high of~0.7μΜ. Excessive ROS are removed by antioxidant systems to maintainredox homeostasis. The imbalance of ROS production alters the intracellular redox homeostasis,causes cellular DNA damage and genomic instability, which contributes to the development andprogression of cancer. But although they have these negative effects, the levels of Reactiveoxygen species (ROS) must be maintained balance inside cells because Reactive oxygen species(ROS) play an important role in the innate immune response against intracellular bacteria andcell growth and survival.To date, researchers have focused mainly on Romo1in humans, and little is known about theexistence of Romo1in Common carp. Reactive oxygen species modulator (Romo1) has beendemonstrated to play important functions in ROS production and cell proliferation, but there isless knowledge available on innate immune system, the immune function of Romo1has onlyfound in the Lampetra japonica.In this paper, we have isolated and sequenced the full-length cDNA of Romo1from thecommon carp by the method of molecular cloning and rapid-amplification of cDNA ends(RACE). The primer pairs used for experience are designed on the basis of the conserved regionof zebrafish compared with other animals. A full length of Romo1cDNA was amplified fromthe liver of common carp (C. carpioL.) using RT-PCR and RACE. The complete cDNA was480bp in length and contained a112bp50-untranslated region (UTR), an open reading frame (ORF)of240bp and a128bp30-UTR with an ATTAAA sequence appeared in the end, which was a common variant of poly(A) signal AATAAA. The ORF of common carp Romo1cDNA encodeda protein of79amino acids. The predicted molecular weight of the Romo1protein was8.34KDaand a theoretical isoelectric point was9.89. In accord with other Romo1s, which contained aReactive mitochondrial oxygen species modulator superfamily (13-79aa). In addition,the Romo1contained a transmembrane region (23-45aa) and included a overlop (21-40aa). Sequencealignment of common carp Romo1with some other known Romo1s was performed at the aminoacid level.The amino acid sequence of common carp Romo1was highly similar to otherRomo1s. It showed a highly similar to other Romo1s and showed a high degree of conservationover a long period of evolution.In order to determine the expression pattern of the Romo1in different tissues, Romo1mRNAwas quantified in a wide range of tissues from normal common carp, including liver, muscle,gonad, systemic immune organs-spleen and head kidney, and mucosal immune organs ortissues-foregut, hindgut, skin and oral epithelial. The results of the Real-time RCR analysisshowed the Romo1gene was expressed in all tissues tested.To elucidate the antibacterial function of common carp Romo1, we analyzed the levels ofRomo1expression in common carp after after i.p. injection with Aeromonas hydrophila. Weobserved that Romo1mRNA was quickly induced in almost all selected tissues of common carp(P<0.05or P<0.01). The upregulated expression level of common carp Romo1in foregut wasmost significant among the immune-related tissues. The upregulated espression level of Romo1in common carp gills was less apparent. After bacterial challenge, the level of Romo1mRNA inthe skin and spleen showed a little change. These results suggest that Romo1may be involved inthe antibacterial immune response and may be an inducible gene associated with anti-bacterialdefence.