| With the global climate warming in rencent years,the life of mussels is widely affected by temperature stress.The effect of temperature stress on the mussel in apoptosis and its mechanism are unclear.Here,trypan blue and neutral red were used to tested the cell viablity and lysosomal stability of Mytilus coruscus and M.galloprovincialis to temperature stress,and the apoptosis of M.galloprovincialis hemocytes was mainly studied by tunel technique and flow cytometry analysis.The full-length cDNA of p38 MAPK,caspase 3/7 were cloned by RT-PCR and RACE-PCR techniques in mussels,Mytilus coruscus and M.galloprovincialis.And their structure and characteristics were analysed.The tissue expression and temporal expression profiles of these genes after temperature stress were detected by Real-time PCR technology.Additionally,caspase 3/7 enzyme activity in the two mussels after temperature stress were analysed.The main results are as follows:(1)The cell viablity of hemocytes of M.coruscus was significantly decreased after 2? 2h?6? 0h?2h and 32? 0h?2h,while 28? stress influenced it slightly.The lysosomal stability of hemocytes of M.coruscus was significantly decreased after S0,S2,R0,R24 of 2? and 6?stress.And it significantly decreased after 28? 0 h and significantly increased after R0 and was highest after R24.And it significantly increased after 32? stress at S0,S2,R0,R24.Similarly,The cell viablity of hemocytes of M.galloprovincialis was significantly decreased after 2?stress at S2 and R24,6? stress at S0 and R24,32? stress at S0 and S2.The lysosomal stability of hemocytes of M.galloprovincialis was significantly decreased after 2? stress at S0,S2,R0,R24,6? stress at S0,R0,R24 and 28? stress at S0,S2,R0.And it significantly increased after28?stress at R24 and 32? stress at S0,S2,R0,R24.In addition,apoptosis hemocytes of M.galloprovincialis were significantly increased by 2? stress at S0 and S2 and 6? stress at S2.And high temperature 28? and 32? stress had no obvious effect on the apoptosis number of M.galloprovincialis hemocytes.(2)The full-length cDNA of M.coruscus caspase 3/7 was of 1203 bp,including a3'-terminal UTR of 182 bp,a 5'-terminal untranslated region(UTR)of 106 bp and an open reading frame(ORF)of 915 bp.Similarly,the full-length cDNA of M.galloprovincialis caspase3/7 was of 1231 bp,including a 3'-terminal UTR of 193 bp,a 5'-terminal untranslated region(UTR)of 120 bp and an open reading frame(ORF)of 918 bp.The M.coruscus caspase 3/7encodes a polypeptide of 304 amino acids and M.galloprovincialis caspase 3/7 encodes a moreamino acid,contained the same casc domain(48-302 aa in M.coruscus,49-303 aa in M.galloprovincialis).The predominant expression of caspase 3/7 was in hemocytes of M.coruscus and the highest expression of caspase 3/7 was in gill tissue of M.galloprovincialis.The caspase3/7 transcript level was significantly increased after 2? R24 and 6? R0 in the gill,2? and 6?stress in hemocytes of M.coruscus.Additionally,the M.coruscus caspase 3/7 enzyme activity significantly increased after 2? stress and significantly decreased after 6?,28? and 32?stress in gill.And it significantly increased after 2?,6?,28? and 32? stress at different degrees in hemocytes of M.coruscus.Different from M.coruscus,the M.galloprovincialis p38 MAPK transcript level significantly decreased after 2?,6?,28? and 32? stress in gill,significantly increased after 6? 2h and 28? 0 h in digestive gland and after 28? 0 h and 32?R0 in hemocytes.The M.galloprovincialis caspase 3/7 enzyme activity significantly increased after 2?,6?,28? and 32? stress in gill,2?,6?,28? stress in hemocytes.And it significantly decreased after 32? stress in hemocytes of M.galloprovincialis.(3)The full-length cDNA of M.coruscus p38-MAPK was of 1502 bp,including a3'-terminal UTR of 144 bp,a 5'-terminal untranslated region(UTR)of 296 bp and an open reading frame(ORF)of 1062 bp.Similarly,the full-length cDNA of M.galloprovincialis p38-MAPK was of 1443 bp,including a 3'-terminal UTR of 231 bp,a 5'-terminal untranslated region(UTR)of 150 bp and an open reading frame(ORF)of 1062 bp.The two mussel p38-MAPKs encode a polypeptide of 353 amino acids,contained the same S_TKc domain(26-310 aa).The predominant expression of p38-MAPK was in hemocytes of M.coruscus and the highest expression of p38-MAPK was in gill tissue of M.galloprovincialis.The p38-MAPK transcript level was significantly increased after 6? stress in the gill and digestive gland of M.coruscus.While it significantly decreased after 2?,6?,28? and 32? stress at different degrees in hemocytes of M.coruscus.Different from M.coruscus,the M.galloprovincialis p38-MAPK transcript level significantly increased after 2? stress in gill,6? 2h and 28? 0h in digestive gland,28? 0h and 32? 2h in hemocytes. |
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