Expression And Functional Analysis Of Prx-4and Serpin Genes From The Mollusk Octopus Ocellatus

Octopus ocellatus is widespread in coastal areas of South and NorthChina. Owing to its rich nutrition, tender and delicious flesh, and rapidgrowth, O. ocellatus is emerging as one of the important economic speciesof octopus on China’s northern coast. However, the farming of O.ocellatus is hindered by a variety of problems, such as low survival rate,poor stress resistance, and unstable seed supply. Thus, this species iscurrently obtained mainly through sea fishing.With dwindling natural resources, how to increase the germplasmquality and survival rate of farmed individuals as well as to improve theexisting mode of farming for O. ocellatus has become the problem to besolved. In this circumstance, studies on the immune system of O. ocellatus,including the cloning and identification of immune-related genes, theanalysis of target gene expression regulation rules, and the exploration oftarget gene functions in immune defense, are of critical importance forbetter understanding of the immune mechanisms, screening of resistance genes, and improvement of disease resistance in O. ocellatus for farmingpractices.In the present study, the encoding genes of a peroxiredoxin-4（Prx-4）gene and a serine protease inhibitor （Serpin） were identified from a cDNAlibrary of O. ocellatus （designated as OoPrx-4and OoSerpin, respectively）using modern molecular biology techniques. The expression patterns ofOoPrx-4and OoSerpin in healthy O. ocellatus tissues as well ashemocytes challenged by Gram-negative Vibrio anguillarum andGram-positive Micrococcus luteus were characterized. Further, thebiological functions of recombinant OoSerpin （designated as rOoSerpin）were evaluated.Prx is a member of the antioxidant proteins superfamily, whichwidely occurs in aerobic organisms and plays a crucial protective roleagainst the toxicity of reactive oxygen species （ROS）. Full-length cDNAof OoPrx-4gene obtained in this study was934bp, which contained a5’-untranslated region （UTR） of19bp, a3’-UTR of177bp with a poly （A）tail, and an open reading frame （ORF） of738bp, encoding a polypeptideof245amino acids. The molecular mass of the deduced amino acidsequence was27.1kDa, with an estimated isoelectric point of6.65. Aminoacid sequence1（M）-19（G） was predicted to be the signal peptide, and51（I）-201（L） was the reductase domain. Multiple sequence alignments revealed that OoPrx-4shared the highest sequence identities withCrassostrea gigas （EKC37672） and Biomphalaria glabrata （ACI42880）Prx-4precursors （78%and73%, respectively）.Quantitative real-time PCR （qRT-PCR） assays were performed toassess mRNA expression of OoPrx-4in various tissues and its temporalexpression in haemocytes challenged by V. anguillarum. OoPrx-4mRNAwas constitutively expressed in all healthy O. ocellatus tissues tested,including mantle, muscle, renal sac, gill, hemocyte, gonad, systemic heart,and hepatopancreas. The highest OoPrx-4mRNA expression level wasdetected in the hepatopancreas, approximately70-fold greater than thelowest level in hemocytes. After V. anguillarum challenge, OoPrx-4mRNA expression in hemocytes was significantly up-regulated by5.7-and28-fold at6and48h, respectively （P <0.01for both comparisons）.The results indicated that OoPrx-4plays an important role in scavengingROS generated during the defense against viral invasion.Serpin is an important member of serine proteinase inhibitors, whichis capable of regulating proteolytic events and is involved in a variety ofphysiological processes. Full-length cDNA of OoSerpin was1735bp,which contained a5’-UTR of214bp, a3’-UTR of282bp, and an ORF of1239bp, encoding a polypeptide of412amino acids. The predictedmolecular weight of the deduced amino acid sequence was46.5kDa with an isoelectric point of8.52. The amino acid sequence1（M）-25（S） waspredicted to be a signal peptide, and49（A）-410（P） was the serpin domain.Multiple sequence alignments revealed that OoSerpin shared the highestsequence identity （37%） with Mus musculus （NP₉41373） and Ixodesscapularis （XP₀02407493） Serpin genes.The expression patterns of OoSerpin in healthy ocellatus tissues aswell as bacteria-challenged hemocytes were assessed by qRT-PCR assays.OoSerpin mRNAof was constitutively expressed in all O. ocellatus tissuestested. The highest level of tissue-specific OoSerpin mRNA expression inO. ocellatus was detected in the hepatopancreas followed by the systemicheart, and the lowest level was in the mantle, approximately760-foldlower than the highest level.After challenged by V. anguillarum, the transcriptional level ofOoSerpin in O. ocellatus hemocytes was significantly up-regulated by6-fold at24h compared with that before challenge （P <0.01）. The highestlevel of O. ocellatus expression was observed at48h post challenge, i.e.,85-fold that before challenge. In the M. luteus-challenged group, O.ocellatus expression level was significantly upregulated at6and12h by4-and3-fold compared with that before challenge. The highest level of O.ocellatus expression was observed at24h post challenge, i.e.,26-fold thatbefore challenge, which declined again at48h post challenge, i.e.,14-fold that before challenge.OoSerpin-encoding region was cloned into the pEASY-E1expressionvector and then transformed into Escherichia coli Transetta（DE3）expression strain. After4h of1%IPTG-induced expression, the proteinproduct was purified and renaturated to obtain rOoSerpin. The inhibitoryeffects of rOoSerpin on trypsin and chymotrypsin activities as well as thegrowth of E. coli were assayed. After0.5h of mixed incubation,960μg ofrOoSerpin respectively inhibited86.5%and55.0%of trypsin （50μg） andchymotrypsin （25μg） activities. Additionally, rOoSerpin significantlyinhibited the growth of E. coli by40.6%in7.5h. The results suggest thatOoSerpin expression can be induced by stimulation of microbialpathogens. The inhibitory effects of rOoSerpin on serine proteases andbacterial growth demonstrate that OoSerpin is involved in the immuneresponse against bacterial infection in mollusk.In conclusion, OoPrx-4and OoSerpin genes are importantcomponents involved in the immune defense response of O. ocellatus.This study provides new insights into the molecular basis and regulationmechanism of immune-related genes in O. ocellatus, thereby contributingto the knowledge of immunology in invertebrate.