杂色鲍DAD1的克隆及在发育、弧菌感染、高温和缺氧下的表达分析

Molecular cloning and expression of defender against cell death1 from Haliotis diversicolor in development, vibrio infection,thermal stress and hypoxia stress

抗细胞凋亡因子(DAD1)是细胞凋亡的负调控因子。在杂色鲍转录组测序的基础上,通过RACE的方法,获得了杂色鲍DAD1基因的全长c DNA,命名为HdDAD1。该序列全长553 bp,开放阅读框339 bp,编码112个氨基酸。编码蛋白具有保守的DAD功能域和3段跨膜区。实时定量PCR结果表明,HdDAD1在杂色鲍所检测组织中均有表达,消化道、鳃、血细胞、粘液腺和肾脏中表达量最高。同样,HdDAD1在发育各阶段均有表达,幼虫阶段表达量最高,原肠胚次之,其他胚胎发育阶段和稚鲍表达量最低。HdDAD1能够响应细菌感染、高温和缺氧应激。在弧菌注射12 h后,表达量显著上升。当水温由最适的25°C上升到28°C时,HdDAD1表达量升高;在31°C持续4和96 h时HdDAD1表达量显著上升。在低氧处理4和96 h时,HdDAD1的表达量也显著上升。

The defender against cell death 1（DAD1） is a regulatory protein to inhibit the apoptosis process. The full-length c DNA of Haliotis diversicolor DAD1（HdDAD1） was cloned by RACE according to DAD1 partial sequence of transcriptome. HdDAD1 was 553 bp, with a 339 bp open reading frame encoding a protein of 112 aa（HdDAD1）. HdDAD1 contains a conserved DAD domain, with 3 transmembrane spans. Quantitative real-time PCR results indicated that HdDAD1 could be detected in all examined tissues, with the highest expression level in digestive tract, gill, hemocyte, mucus gland and kidney. There was also expression of HdDAD1 in different developmental stages. HdDAD1 expression level of larva stages was the highest, that of gastrula stage was middle,that of other stages was the lowest. HdDAD1 could respond bacterial challenge/thermal/hypoxia stress. There was significant up-regulation of HdDAD1 at 12 h after Vibrio parahaemolyticus challenge. HdDAD1 was significantly up-regulated, when water temperature rose from 25 to 28 °C. The expression of HdDAD1 also significantly increased, when water temperature was 31 °C for 4 and 96 h. At 4 and 96 h post hypoxia stress, HdDAD1 significantly increased, too.