利用cDNA末端快速扩增技术(rapid amplification of cDNA ends, RACE)克隆了大珠母贝CCT-eta cDNA全长。利用多种生物信息学软件对大珠母贝CCT-eta cDNA和预测的氨基酸序列进行了分析, 并采用实时荧光定量PCR对其组织表达谱和冷应激下的表达模式进行了分析。结果显示, 大珠母贝CCT-eta cDNA全长为1895bp, 其中开放阅读框(ORF)为1632bp, 编码543个氨基酸残基, 含有CCT家族的3个签名序列: 37RTTLGPRGMDKLI49、58ISNDGATILKTLDIVHP74、86QDAEVGDGT94, 具有3个保守的功能结构域: 赤道结构域、顶端结构域和中间结构域。氨基酸同源序列比对结果显示, 大珠母贝CCT-eta与其他物种的同源性较高, 与长牡蛎(Crassostrea gigas)、斑马鱼(Danio rerio)、人(Homo sapiens)的同源性分别为83.2%、80.1%、79.3%。CCT-eta基因在大珠母贝各组织中均有表达, 但在闭壳肌中表达水平最高, 而在心脏和肝胰腺中表达很低; 冷应激后, 大珠母贝各组织中CCT-eta表达显著上调, 但不同组织的冷应激响应模式不同, 闭壳肌响应速度较慢, 而心脏和肝胰腺响应速度较快, 提示冷应激时CCT-eta对保护大珠母贝肝胰腺和心脏等内脏组织具有重要作用。
毛艾涛
,
唐啸尘
,
刘志刚
. 大珠母贝CCT-eta基因cDNA克隆及其冷应激表达[J]. 热带海洋学报, 2015
, 34(6)
: 57
-63
.
DOI: 10.11978/2015021
The cDNA sequence of Pinctada maxima CCT-eta (PmCCT-eta) was cloned by rapid amplification of cDNA ends method (RACE) and bioinformatics analysis of PmCCT-eta was conducted. The distribution of PmCCT-eta in various tissues and the induced expression of PmCCT-eta under cold stress in Pinctada maxima were studied by real-time PCR. The results showed that the full length of PmCCT-eta cDNA was 1895 bp, which contained a 1632 bp ORF encoding 543 aa. Three CCT-eta signature sequences (37RTTLGPRGMDKLI49, 58ISNDGATILKTLDIVHP74, 86QDAEVGDGT94) and three functional structure domains (Equatorial domain, Apical domain, Intermediate domain) were found in the deduced protein of PmCCT-eta. Homology analysis indicated that the deduced protein of PmCCT-eta had 83.2%, 80.1% and 79.3% similarity with CCT-eta of Crassostrea gigas, Danio rerio and Homo sapiens, respectively. PmCCT-eta mRNA expression in all tissues was observed, with highest expression in adductor muscle and lower expression in heart and hepatopancreas. The PmCCT-eta mRNA expression was up-regulated in adductor muscle, heart and hepatopancreas of Pinctada maxima under cold stress condition. However, different tissues had distinct response patterns; heart and hepatopancreas responded faster than adductor muscle, suggesting that CCT-eta plays an important role in resisting cold stress in internal organs such as heart and hepatopancreas.
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