魁蚶(Scapharca broughtonii)热休克蛋白90(HSP90)基因的克隆及转录表达分析

摘要

热休克蛋白90(HSP90)作为一种研究的常规免疫基因,在许多物种都有过报道.本研究从构建的魁蚶转录组文库中筛选得到的HSP90基因部分序列为基础,通过RACE技术获得其cDNA全长序列(命名为SbHSP90),以期明确魁蚶HSP90基因的结构特征、组织分布及其对病原菌刺激的免疫变化规律.序列和结构分析表明,该cDNA全长2707bp,编码一个由728个氨基酸组成的多肽,该多肽含有HSP90家族共有的5个签名序列,C端高度保守的MEEVD短肽序列和ATPase结构域;预测蛋白的分子量(Mw)为83.72kDa,理论等电点(pI)为4.85;预测该蛋白无信号肽,具有4个糖基化位点.同源性及系统分析表明,SbHSP90基因与软体动物的HSP90相似性达到83％以上,其中与长牡蛎(Crassostrea gigas)和海湾扇贝(Argopecten irradians)相似度最高达86％,与甲壳动物HSP90的相似度都在81％左右,与脊椎动物HSP90-α和HSP90-β的同源性都很接近.实时荧光定量PCR(qRT-PCR)结果表明:SbHSP90 mRNA在魁蚶血细胞、斧足、鳃、外套膜、闭壳肌和肝胰腺和中均有表达,斧足中的表达量相对较高,而在肝胰腺中的表达量则相对较低;注射鳗弧菌后,相对于对照组,SbHSP90基因在所检测的每个组织中mRNA水平上的表达量都显著上调(P＜0.01),而且具有显著的时间依赖性和瞬时表达趋势.%As a molecular chaperone,HSP90 plays pivotal roles in protein process in a normal condition.However,it could be involved in the immune response to pathogens invasion in other abnormal conditions.In the present study,we cloned the full length cDNA of heat shock protein 90 from ark shell Scapharca broughtonii (designated SbHSP90) by rapid amplification of cDNA ends (RACE) mean with partial cDNA sequence selected from the trancriptome library.The full length cDNA of SbHSP90 was 2707bp,containing 195bp of 5'untranslated region (UTR) of,325bp of 3'-UTR,and 2187bp of open reading frame (ORF) encoding a peptide with 728 amino acids.It owned five signature sequences of HSP90 family,an ATPase domain,and a highly conserved short peptide MEEDV in the C-terminus.The predicted molecular weight (Mw) was 83.72kDa and the theoretical isoelectric point (pI) was 4.85.There was no a signal peptide in the N-terminus,and four glycosylation sites were predicted in the deduced amino acids.The sequence analysis showed that the identity of SbHSP90 shared with mollusks was over 83％,crustacean was about 81％,and the identity with HSP90-α was similar to HSP90-β from vertebrates.Real-time quantitative PCR (qRT-PCR) analysis detected that SbHSP90 expressed in all examined tissues of S.broughtonii,including hemocytes,foot,adductor muscle,mantle,gill,and hepatopancreas,and the relative highest expression level was observed in foot.After challenged with Vibrio anguillarum,the relative mRNA expression level of SbHSP90 was up-regulated significantly (P＜0.01),the times to reach maximum expression level and return to initial level were differ in different tissues.So SbHSP90 showed obvious time dependence and instantaneous expression trends.All these data suggested that SbHSP90 played a key role in innate immune process of ark shells.