大珠母贝CCT-eta基因cDNA克隆及其冷应激表达  

cDNA cloning of CCT-eta gene from Pinctada maxima and expression analysis under cold stress condition

作　　者：毛艾涛 唐啸尘[1] 刘志刚[1] 

机构地区：[1]广东海洋大学水产学院,广东湛江524088

出　　处：《热带海洋学报》2015年第6期57-63,共7页Journal of Tropical Oceanography

基　　金：广东省海洋渔业科技推广专项科技攻关与研发项目(A201008A01;A201201A06;A201208A07)

摘　　要：利用c DNA末端快速扩增技术(rapid amplification of c DNA ends,RACE)克隆了大珠母贝CCT-eta c DNA全长。利用多种生物信息学软件对大珠母贝CCT-eta c DNA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 c DNA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。The cDNA sequence ofPinctada maxima CCT-eta (PmCCT-eta) was cloned by rapid amplification of cDNA ends method (RACE) and bioinformatics analysis ofPmCCT-eta was conducted. The distribution ofPmCCT-eta in various tissues and the induced expression ofPmCCT-eta under cold stress inPinctada maxima were studied by real-time PCR. The results showed that the full length ofPmCCT-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 andHomo sapiens,respectively.PmCCT-eta mRNA expression in all tissues was observed, with highest expression in adductor muscle and lower expression in heart and hepatopancreas. ThePmCCT-eta mRNA expression was up-regulated in adductor muscle, heart and hepatopancreas ofPinctada 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.

关 键 词：大珠母贝 CCT-eta CDNA 组织表达 冷应激 

分 类 号：Q785[生物学—分子生物学] Q786