拟穴青蟹GRP78基因的克隆与应激表达

葡萄糖调节蛋白78(glucose regulated protein 78 ku,GRP78)是热休克蛋白70家族成员之一,在调节蛋白质折叠和维持内质网稳态过程中起着分子伴 侣作用。采用RT-PCR、RACE等技术,首次从拟穴青蟹获得了GRP78的cDNA全长序列。该序列全长2 284 bp,开放阅读框(ORF)为1 962 bp,编码653个氨基酸残基。 同源分析显示,该基因编码的蛋白含有HSP70家族的签名序列,C末端为内质网蛋白滞留信号KDEL,与其他物种具有很高相似性。实时荧光定量PCR结果表明,GRP78 基因在拟穴青蟹多个组织中均有表达。第一期仔蟹在不同的温度和盐度下暴露12 h后,GRP78基因表达量随环境温度升高而增加;在高盐(30)条件下GRP78表达量 较高,进而推测拟穴青蟹GRP78参与蛋白质折叠和环境胁迫的应答。

Cloning and stress expression analysis of glucose regulated protein 78 ku(GRP78)in mud crab(Scylla paramamosain)

Glucose regulated protein 78 ku(GRP78)was one member of heat shock protein 70(HSP70)family.As a molecular chaperone,GRP78 plays important roles in regulating the protein folding and maintaining the stability of endoplasmic reticulum.In this study,we firs cloned the full-length cDNA sequence of GRP78 from mud crab,Scylla paramamosain using RT-PCR and RACE.The full-lengty of GRP78 cDNA is 2 284 bp with 1 962 bp open reading frame encoding 653 amino acids.The homologous analysis indicated that the deduced amino acids contained the signature sequences of HSP70 and the endoplasmic reticulum retention sequence in the C-terminal domain.The deduced amino acid sequence shared high identity with previously reported GRP78s.RT-PCR indicated that GRP78 was expressed in various tissues.When S.paramamosain juveniles exposed to different temperatures and salinities after 12 hours,the expression of GRP78 gene increased with the rise of environmental temperature and a high expression of GRP78 was detected in high salinity(30).So we infer that GRP78 in S.paramamosain might participate in protein folding and the response to environmental stress.