Molecular And Acute Temperature Stress Response Characterizations Of Some Apoptosis-Related Genes In Two Mussels,Mytilus Coruscus And Mytilus Galloprovincialis

Mussels are important species living in the intertidal zone.In recent years,with global climate warming,the distributions of different mussel species have changed.The differences of physiology traits response to temperature stress of two mussels may be closely related with the change of distribution ranges.However,the effect of temperature stress on different mussel species and their response mechanism to temperature stress are poor understood.In the present study,comet assay was used to detect the DNA damage in hemocytes of Mytilus coruscus and Mytilus galloprovincialis after temperature stress.The full length cDNA sequences of caspase-8and Rel were cloned from M.coruscus and M.galloprovincialis and Bcl-2,Bax and Cytochrome c were cloned from M.coruscus by RT-PCR and RACE-PCR,then their structure and characteristics were analysed.The tissue expression and temporal expression profiles of these genes after temperature stress were detected by quantitative real-time PCR.Additionally,caspase-8 enzyme activity in the two mussels after temperature stress was analysed.The main results are as follows:（1）Single-stranded and double-stranded DNA damage were detected in the hemocytes of M.coruscus and M.galloprovincialis.The type and extent of DNA damage are time and temperature dependent.M.galloprovincialis hemocytes single-stranded DNA damage more sensitive to low temperature stress,and the extent of DNA damage in M.coruscus significangtly increased after high and low temperature stress.（2）The full-length cDNA of M.coruscus caspase-8 was 1884 bp,including a 1506bp open reading frame（ORF）,which encoded 501 amino acids.Similarly,the full-length cDNA of M.galloprovincialis caspase-8 was 1775 bp,including a 1488 bp open reading frame（ORF）,which encoded 495 amino acids.The two mussels caspase-8 contained the same DED,CASPASE_P20and CASPASE_P10 domain.The caspase family cysteine active site（KPKLFFIQACQG）was present 348-359 and 345-356,respectively.Quantitative real-time PCR（qRT-PCR）analysis indicated a broad expression of caspase-8 in most detected tissues,with the predominant expression in mantle and the weakest expression in digestive gland of M.coruscus whereas the highest expression in digestive gland and the weakest expression in muscle of M.galloprovincialis.After 2℃stress,caspase-8 enzyme activity showed up-regulation in gill and hemocytes of M.coruscus and in gill of M.galloprovincialis;after 6℃stress,caspase-8expression levels showed up-regulation in gill and hemocytes of M.galloprovincialis,caspase-8enzyme activity showed up-regulation in gill and hemocytes of M.coruscus and M.galloprovincialis;after 28℃stress,caspase-8 expression levels showed up-regulation in gill and hemocytes of M.coruscus and gill of M.galloprovincialis,caspase-8 enzyme activity showed up-regulation in gill of M.coruscus and M.galloprovincialis;after 32℃stress,caspase-8expression levels showed up-regulation in gill and hemocytes of M.coruscus and M.galloprovincialis,caspase-8 enzyme activity showed up-regulation in gill of M.coruscus and in gill and hemocytes of M.galloprovincialis.（3）The full-length cDNA of M.coruscus Rel was 2981 bp,including a 1788 bp open reading frame（ORF）,which encoded 595 amino acids.Similarly,the full-length cDNA of M.galloprovincialis Rel was 2985 bp,including a 1794 bp open reading frame（ORF）,which encoded 597 amino acids.The two mussels Rel contained the same RHD and IPT domain.The Rel protein signature FRYXCE was present 105-110 and 107-112,respectively.qRT-PCR analysis indicated a ubiquitous expression of Rel in most detected tissues,with the predominant expression in mantle and the weakest expression in hemocytes of M.coruscus whereas the highest expression in gill and the weakest expression in digestive gland of M.galloprovincialis.After 2℃stress,Rel expression levels showed up-regulation in gill of M.coruscus;after 6℃stress,Rel expression levels showed up-regulation in gill of M.coruscus and M.galloprovincialis;after 28℃stress,Rel expression levels showed up-regulation in hemocytes of M.coruscus and in gill of M.galloprovincialis;after 32℃stress,Rel expression levels showed up-regulation in gill and hemocytes of M.coruscus and M.galloprovincialis.（4）The full-length cDNA of M.coruscus Bcl-2 was 784 bp,including a 558 bp open reading frame（ORF）,which encoded 185 amino acids.The Bcl-2 contained the BH4₂ and BCL2_FAMILY domain.Semiquantitative PCR analysis indicated a broad expression of Bcl-2 in most detected tissues,with the predominant expression in gonad and gill and the weakest expression in hemocytes of M.coruscus and M.galloprovincialis.After 2℃stress,Bcl-2expression levels showed up-regulation in hemocytes of M.coruscus;after 6℃and 28℃stress,Bcl-2 expression levels showed up-regulation in gill and hemocytes of M.coruscus and M.galloprovincialis;after 32℃stress,Bcl-2 expression levels showed up-regulation in hemocytes of M.coruscus and in gill and hemocytes of M.galloprovincialis.（5）The full-length cDNA of M.coruscus Bax was 766 bp,including a 630 bp open reading frame（ORF）,which encoded 209 amino acids.The Bax contained a BCL2_FAMILY domain.Semiquantitative PCR analysis indicated a ubiquitous expression of Bax in most detected tissues,with the predominant expression in digestive gland and muscle and the weakest expression in gill of M.coruscus whereas the highest expression in mantle and muscle and the weakest expression in hemocytes of M.galloprovincialis..After 2℃stress,Bax expression levels showed up-regulation in gill and hemocytes of M.coruscus and in hemocytes of M.galloprovincialis;after 6℃and 28℃stress,Bax expression levels showed up-regulation in gill and hemocytes of M.coruscus and M.galloprovincialis;after 32℃stress,Bcl-2 expression levels showed up-regulation in hemocytes of M.coruscus and in gill and hemocytes of M.galloprovincialis.（6）The full-length cDNA of M.coruscus Cytochrome C was 637 bp,including a 333 bp open reading frame（ORF）,which encoded 110 amino acids.The Cyto C contained the CYTC domain.Cys²⁰ and Cys²³ were covalently bound sites to heme;His²⁴ was a ligand binding site to iron ion.qRT-PCR analysis indicated a ubiquitous expression of Cyto C in most detected tissues,with the predominant expression in gonad and mantle and the weakest expression in hemocytes of M.coruscus whereas the highest expression in mantle and the weakest expression in digestive gland of M.galloprovincialis.After 2℃stress,Cyto C expression levels showed up-regulation in gill of M.coruscus and M.galloprovincialis;after 6℃stress,Cyto C expression levels showed up-regulation in gill and hemocytes of M.galloprovincialis;after 28℃stress,Cyto C expression levels showed up-regulation in gill and hemocytes of M.coruscus and M.galloprovincialis.