| Pinctada fucata martensii is a widespread species involve in marine pearl aquaculture.Pearl production via nucleus implantation or grafting operation has gained much attention worldwide with P.fucata martensii being the commercially cultured species in China,producing more than 90% of pearls.We have developed red,yellow,black,white coloured lines by family and mass selection.Shell colours such as red,yellow,black,white and brown(commonest)were observed to exhibit different growth and reproductive ability.Currently the fifth generation line has been established and it has been observed that among the shell coloured stocks,the black shell coloured stock exhibit higher survival ability after nucleus implantation.In the project,we compared growth,pearl production performance and the age related immune and antioxidant capacity of black coloured line and control group and also ascertain their immune response to grafting.1.Survival,retention rate and immunity of the black shell colored and control stocks of pearl oyster P.fucata martensii after grafting operationIn this study,total carotenoid content(TCC)of the black shell colored line(BG)and the control group(CG)were compared.The BG was subjected to a grafting experiment.Survival and retention rates,immunity and antioxidant capacity of BL were compared relative to CG at different times after the grafting operation.The results showed that BG had significantly larger TCC than CG(P < 0.05).BG had significantly higher survival and retention rates than CG on days 7,30 and 360 after grafting(P<0.05).On days 360,BG had significantly larger pearl thickness than CG(P<0.05).BG had higher ACP,AKP,SOD,CAT,TAOC and LZ activities than the CG on 0h,12 h,1d,3d,5d,7d and 30 d after grafting.BG had expression levels of Fascin,SOD,CDK-7,CDAP-1,IRAK-1,α2m,GST-1,TRAF-3 and Caspase-2 m RNA than CG.The results suggested that BG had higher immune competence and pearl production performances,which is promising to improve pearl quality and production.2.A Comparison of pearl productions performance,enzymatic activity and immune related gene expression of different aged pearl oyster P.fucata martensiiThis study evaluated influences of age on pearl production performance and innate immune response of pearl oyster P.fucata martensii.Two groups(A,1.5 years aged and B,2.5 years aged)were subjected for nucleus insertion operation.The activities of immune and antioxidant enzymes and expression levels of immune related genes were evaluated between the two groups 3 days after nucleus insertion operation.Survival,retention rate,and biomineralization capacity of the groups were examined at harvest of pearls.The results showed that activities of alkaline phosphatase(AKP),acid phosphatase(ACP),superoxide dismutase(SOD),catalase(CAT),and glutathione peroxidase(GPx)and expression of SOD,GPx,Heat Shock Proteins(HSPs)70 and HSP 90 genes were significantly higher in A group than in B group 3 days after the nucleus insertion operation(P < 0.05).The A group had significantly higher survival rates both on 3,7 and 30 days and at harvest of pearls than B group.The A group had significantly higher retention rate and pearl thickness relative to B group at harvest of pearls.The expression levels of biomineralization genes nacrein and pearlin were significantly higher in group A compared to the group B(P < 0.05)except piff-177.Collectively these results suggested that 1.5 year old pearl oysters have a better pearl production performance and innate immune response to nuclei insertion operation,which is promising to improve pearl quality by sampling these young pearl oysters as host and donor oysters.3.Characterization of thioredoxin-Like protein-5 and its differential expression in the black colored selected line of P.fucata martensii to grafting operationThioredoxin-like protein 5(Trxlp-5)is a thioredoxin isoform associated with cellular redox homeostasis through the activity of thiol-disulfide reductase.In our study,Trxlp-5 was identified and characterized in the pearl oyster P.fucata martensii.The expression of Pm Trxlp-5 was detected in response to poly(I:C)and LPS stimulation.The differences in Pm Trxlp-5 expression were evaluated between the black colored selected line and the control stock after grafting operation.The open reading frame(ORF)consisted of 1167 bp encoding a 388 amino acid,5′-UTR of 41 bp and a 3′-UTR of 846 bp.Pm Trxlp-5 exhibited a conserved WCXXC functional motif similar to thioredoxins from other species.Tissue analysis showcased the highest relative m RNA expressions of Pm Trxlp-5 in the haemocytes.Interestingly,after the grafting operation,m RNA expression of Pm Trxlp-5 in the blood was differentially expressed post grafting with a peak 6h after grafting suggesting the high involvement of the gene in immune response in the early stage after grafting.The BS group had significantly higher expression than the CG group at 24 hrs,6d and 30 d after grafting operation.Pm Trxlp-5 also showed a wave-like pattern in m RNA expression after bacterial endotoxin lipopolysaccharides(LPS)and viral mimic polyinosinic: polycytidylic acid(poly I:C).These results from our study showed that Pm Trxlp-5 plays an essential function in cellular redox homeostasis and immune response against grafting operation and pathogenic infections.4.Characterization of cyclin dependent Kinase-7 and its differential expression in the black colored selected line of P.fucata martensii to grafting operationCyclin dependent kinase-7(Cdk-7)is a protein kinase involved in cell cycle,cell differentiation and proliferation,apoptosis and inflammatory response.In this study,the full length c DNA sequence of Pm Cdk-7 was characterized in P.fucata martensii.A full length sequence of 1473 bp with an open reading frame(ORF)of 915 bp encoding a 304 aa,5′-UTR of 58 bp and a 3′-UTR of 500 bp was observed in Pm Cdk-7.The deduced amino acid sequence of Pm Cdk-7 contained Serine/Threonine protein kinases,catalytic(S＿TKc)domain with a protein kinases ATP-binding region signature(14-38aa)and the serine/Threonine protein kinases activesite signature(129-141aa)within the domain.Tissue distribution analysis revealed a high relative m RNA expression of Pm Cdk-7 in the haemocytes.Interestingly,after the grafting operation,m RNA expression of Pm Cdk-7 in the blood was differentially expressed among the studied groups;the black shell colored selected line(BS)and the control group(CG).Post grafting,BS exhibited high expression of Pm Cdk-7 at all-time points than CG with a significance and peak at 3d suggesting a heightened level of DNA replication and inflammatory response.After bacterial endotoxin lipopolysaccharides(LPS)and viral mimic polyinosinic: polycytidylic acid(poly I:C)stimulation,Pm Cdk-7 relative expression was highly expressed at 24 h and down-regulated at 12 h for the poly I:C group and highly at 18 h and lowly at 24 h in the LPS group.The results from our study showed that Pm Cdk-7 plays a vital role in the cell cycle by aiding DNA replication and also aid response to inflammations generated due to the incision from the grafting operation and long exposure to immune-stimulants(pathogens). |
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