CAJ | 학술논문

利用3′RACE技术,对高通量转录组测序所得三角帆蚌HSP60基因(hcHSP60)长片段(2629 bp)进行了3￠末端克隆,经拼接得到hcHSP60 cDNA全长序列。采用多种分子生物学软件对hcHSP60 cDNA全长序列进行了特征分析,并采用 RT-PCR 技术检测了其组织分布及经冷热应激后的表达变化。结果显示, hcHSP60 cDNA全长为2807 bp,其中开放阅读框为1707 bp,编码568个氨基酸,预测分子量大小为61.04 ku, pH 7.0时的理论等电点为5.63。序列中不存在信号肽与跨膜结构。氨基酸序列保守性分析表明, hcHSP60氨基酸序列与光滑双脐螺HSP60同源性最高(达82%),而与牙鲆、白云金丝鱼HSP60的同源性最低(为75%)。RT-PCR检测结果表明, hcHSP60在肝胰腺中的表达水平最高,而在血液中的表达水平最低。30℃与35℃水温处理三角帆蚌4h后,各组织中hcHSP60表达水平明显上升,表明hcHSP60可能在三角帆蚌耐热应激反应中起着重要作用。
이용3′RACE기술,대고통량전록조측서소득삼각범방HSP60기인(hcHSP60)장편단(2629 bp)진행료3￠말단극륭,경병접득도hcHSP60 cDNA전장서렬。채용다충분자생물학연건대hcHSP60 cDNA전장서렬진행료특정분석,병채용 RT-PCR 기술검측료기조직분포급경랭열응격후적표체변화。결과현시, hcHSP60 cDNA전장위2807 bp,기중개방열독광위1707 bp,편마568개안기산,예측분자량대소위61.04 ku, pH 7.0시적이론등전점위5.63。서렬중불존재신호태여과막결구。안기산서렬보수성분석표명, hcHSP60안기산서렬여광활쌍제라HSP60동원성최고(체82%),이여아평、백운금사어HSP60적동원성최저(위75%)。RT-PCR검측결과표명, hcHSP60재간이선중적표체수평최고,이재혈액중적표체수평최저。30℃여35℃수온처리삼각범방4h후,각조직중hcHSP60표체수평명현상승,표명hcHSP60가능재삼각범방내열응격반응중기착중요작용。
Heat shock protein 60 (HSP60) is an important molecular chaperone protein that mainly exists in the mito-chondria of organism. In the present study, the cDNA sequence of Hyriopsis cumingii HSP60 (hcHSP60) was cloned by 3′rapid amplification of cDNA ends methods (3′-RACE) based on a long chain sequence of hcHSP60 (2629 bp) and was determined by high flux sequencing for transcriptome of H. cumingii blood cells, and its expression in the different tis-sues was detected by using reverse transcription-polymerase chain reaction (RT-PCR). Results showed that the full-length cDNA of hcHSP60 was 2807 bp that contains an open reading frame of 1707 bp, encoding a protein of 568 amino acid residues with 61.04 ku of predicted molecular weight and 5.63 of the theoretical isoelectric point, which was predicted to have no signal peptide and transmembrane helices. The deduced amino acid sequence of hcHSP60 shares the highest identity (82%) with HSP60 of Biomphalaria glabrata, and the phylogenetic analysis demonstrated that they were clustered in a same clade. The results of RT-PCR indicated that hcHSP60 was constitutively expressed in all 5 examined tissues of H. cumingii with the highest expression in hepatopancreas. The expression of hcHSP60 in all de-tected tissues was up-regulated obviously by higher water temperature, suggesting that it may play an important role in the stress response against heat.