Effects Of Carotenoids Content And SCD MRNA Level On Resistance Of The Pearl Oyster,Pinctada Fucata To High Temperature Stress

Carotenoids are a class of natural antioxidants widely found in aquatic animals,and they have significant effects on the growth,survival,and immunity of these organisms.The pearl oysters,Pinctada fucata,are economically important shellfish for the production of seawater pearls in China,which were mainly cultured on the Guangxi,Guangdong,and Hainan coasts.In recent years,farming of P.fucata has been facing severe challenges owing to dramatic environmental changes such as rising temperatures.To investigate what roles of carotenoids and SCD gene play in high temperature resistance,a research about antioxidant immune reponses to high temprature stress inclouding Superoxide dismutase(SOD),Catalase(CAT)and Malondialdehyde(MDA),and changes of Total antioxidant capacity(TAC)and Total carotenoids content(TCC)in pearl oyster P.fucata with different carotenoids content were conducted.Secondly,the full length of PfSCD gene cDNA from P.fucata was cloned for the first time,then PfSCD gene tissue-specifi expression and its correlation with TAC and TCC of P.fucata under high temprature stress were investigated,which preliminary confirmed that carotenoids played a significant positive role in the high temperature resistance of P.fucata and provided a new clue for breeding high temperature resistance strains of P.fucata.The research results were as follows:1.Immune response of selected Pinctada fucata families with different carotenoids contents to high temperature stressThe results indicated that the survival rate(SR)of P.fucata decreased significantly with increase in temperature from 26 °C to 34 °C and with the decrease of TCC(P<0.05);when the TCC was higher,the SR tended to be higher.TCC and TAC decreased significantly at 30 °C with increasing stress time(P<0.05).Correlation analysis indicated that TAC was positively and linearly correlated with TCC,and SR was S-type correlated with TCC and TAC(P<0.01).Immune analysis indicated that levels of SOD,CAT and MDA in selected families(with higher TCC)under temperature stress(at 30 °C)were generally significantly lower than in the control group(with lowest TCC)and from 0 to 96 h,the levels of each of these substances varied significantly(P<0.05).Levels of SOD,CAT,and MDA within each family first rose from 0 to 3 h,then decreased to their lowest point after 24 h,and then rose again to their highest levels at 96 h.When TCC was higher,the levels of SOD,CAT,and MDA tended to be lower.These findings indicated that carotenoids play an important role in improving survival rates of P.fucata under high temperature stress by enhancing animals' antioxidant system,and could serve as an index for breeding stress-resistant lines in selective breeding practices.2.Molecular characterization and phylogenetic analysis of SCD gene in Pincatda fucataUsing gene cloning,the SCD gene in P.fucata was obtained and named as PfSCD(Gen Bank accession number: KY849348)and its cDNA full length consists of 1576 nucleotides with an 945 bp open reading frame(ORF)encoding 315 amino acid residues,and 442 bp 3' UTR and 189 bp 5' UTR.The amino acid sequence had four transmembrane domains(TMs,32-53,65-83,177-197,203-224 aa)and three conserved histidine-box motifs(HMs,80-85,117-121,258-262 aa),consistent with other species' SCD sequences as indicated by multiple sequences alignment(MAS).In addition,there were five functional domains inclouding three Fatty acid domains : Fatty acid hydroxylase superfamily(FA_hydroxylase)and Fatty acid desaturase(FAD),a Bipartite nuclear localization signal profile(NLS_BP)and a Protein of unknown function(DUF321)located at 36-122,61-301,254-268,195-215,296-310 aa in the sequence.Phylogenetic analysis indicated that P.fucata PfSCD was clustered together with Crassostrea gigas SCD at first and then other molluscs such as Hyriopsis cumingii,Patinopecten yessoensis and Octopus vulgaris and compairing to others,PfSCD had a more close genetic relationship with C.gigas and other molluscs.PfSCD mRNA real-time expression levels of P.fucata were analyzed by real-time qRT-PCR(quantitative reverse transcription PCR).The results showed that there was a development stages and tissues specific fashion with the highest expression levels in metamorphosis larve stage and hepatopancreas,and the lowest expression levels in trochophore larve stage and foot.3.PfSCD mRNA tissues and development stages specific expression,and correlation between total carotenoids content and total antioxidant capacity in Pincatda fucataPfSCD mRNA real-time expression levels of P.fucata in tissues of hepatopancreas,gonad,and adductor were signifiacantly higher than that of mantle,gill and foot of both orange group(test)and white group(control)(P<0.05)with or without high temperature stress.Besides,the expression levels of orange group were remarkably higher than that of white group with the same tissues under same high temperature stress time(P<0.01).During the stress time from 0 to 96 h,the expression levels in different tissues of orange group and white group presented a trend of rise at first and then of fall.However,the expression levels of orange group reached maximum after 12 h but 3 h for the white group.Moreover,the TAC and TCC in hepatopancreas,gonad,and adductor of orange group and white group presented a saddle pattern meaning that TAC and TCC first decline,and then increase,and then decline,while other tissues constantly decrease.The TAC and TCC of orange group were remarkably higher than that of white group with the same tissues under same high temperature stress time(P<0.01).Further,there were significantly positive correlations between TAC and TCC in the pearl oysters(P<0.05).The present results suggested that carotenoids play essential roles in enhancing the pearl oysters' resistence to high temperature stress,which provided a new clue for breeding new P.fucata strains.