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采用RACE技术克隆获得团头鲂caspase3基因cDNA序列全长,并对该序列进行分析。结果显示,团头鲂caspase3基因全长为2 077 bp,开放阅读框长837 bp,5’非编码区长155 bp,3’非编码区长1 085 bp,命名为Ma Casp3。推测该基因编码278个氨基酸,预测分子量为30.92 ku,理论等电点为6.15。同源性和系统进化分析发现,Ma Casp3基因与斑马鱼(Danio rerio)caspase3氨基酸相似性最高,与其他节肢动物caspase3家族基因聚为一类。荧光定量RT-PCR结果显示,Ma Casp3基因在肝脏中的相对表达量最高,在肌肉中表达量最低。与常温对照组相比,高温胁迫7 d后该基因在团头鲂肝脏中的表达量显著升高,表明Ma Casp3基因可能参与团头鲂高温胁迫的应答并引发细胞凋亡级联反应。此外,高温胁迫7 d与胁迫前比较团头鲂幼鱼肝脏组织呈现肝细胞肿大、混浊变性、细胞核溶解、肝细胞空泡化现象,因此实际生产中应密切关注养殖水体水温变化以减少温度应激导致的组织损伤。
The full length cDNA of caspase3 gene was cloned by RACE technique and the sequence was analyzed. The results showed that the full length of caspase3 gene was 2 077 bp, the open reading frame was 837 bp, the 5 ’non-coding region was 155 bp long, and the 3’ non-coding region was 1 085 bp, which was named Ma Casp3. It was speculated that the gene encoding 278 amino acids, the predicted molecular weight of 30.92 ku, the theoretical isoelectric point of 6.15. Homology and phylogenetic analysis showed that the Caspase3 amino acid sequence of Ma Casp3 gene shared the highest similarity with that of Danio rerio and clustered with other arthropod caspase3 family genes. Fluorescent quantitative RT-PCR results showed that Ma Casp3 gene had the highest relative expression in liver and the lowest in muscle. Compared with the normal temperature control group, the expression of Ma Casp3 gene was significantly increased in the liver of the B group after 7 days of high temperature stress, indicating that Ma Casp3 may be involved in the response to high temperature stress and trigger the cascade of apoptosis. In addition, liver tissue swelling, turbidity degeneration, nuclear lysis and hepatocyte vacuolization appeared in the liver tissue of 7-day-old juvenile fish under high temperature stress compared with those before stress. Therefore, the actual production should pay close attention to the change of water temperature in breeding water to reduce the temperature Stress-induced tissue damage.