马氏珠母贝多聚泛素基因重复单元及3'端非编码区序列的克隆与表达分析

Cloning and Expression Analysis of Repeat Unit and 3′ Untranslated Region cDNA Sequences of Polyubiquitin from Pinctada fucata

在构建马氏珠母贝(Pinctada fucata)血淋巴抑制性差减文库的基础上,应用cDNA末端快速扩增(RACE)技术成功克隆马氏珠母贝多聚泛素基因(PfUb)最后一个重复单元及3'端非编码区(UTR)序列。序列长453 bp,其中3′UTR 219 bp;开放阅读框(ORF)234 bp,编码77个氨基酸残基;PfUb重复单元含有7个Lys和1个76Gly泛素基因功能位点。实时荧光定量PCR分析表明,PfUb基因在马氏珠母贝足、外套膜、鳃、闭壳肌、心脏、消化腺、血淋巴7个组织中均有表达,溶藻弧菌(Vibrio alginolyticus)能够诱导马氏珠母贝各组织中PfUb基因的表达上调,其中以鳃和血淋巴中的上调表达最为明显;马氏珠母贝血淋巴中,PfUb基因在溶藻弧菌感染2 h后表达量开始上调,并在感染8 h后达到最大。

Based on the construction of a suppression subtraction hybridization（SSH） library from haemocyte,the repeat unit and 3′ untranslated region（UTR） cDNA sequences of polyubiquitin from Pinctada fucata（designated PfUb） were cloned by rapid amplification of cDNA ends（RACE） technique.The sequence was 453 bp,consisting of a 3′ UTR of 219 bp and an open reading frame（ORF） of 234 bp encoding a polypeptide of 77 amino acids.Similar to other polyubiquitin,PfUb contained 8 functional sites,including 6Lys,11Lys,27Lys,29Lys,33Lys,48Lys,63Lys and 76Gly in the repeat unit.Real-time PCR analyses indicated that PfUb mRNA was ubiquitously expressed in all tested tissues,and its expression was increased,especially in gill and haemocyte,after Vibrio alginolyticus challenge.The temporal expression of PfUb mRNA in haemocytes challenged by V.alginolyticus was time-dependent.It began to function at 2 h post-challenge and reached the maximum level at 8 h post-challenge.