三角帆蚌热休克蛋白70基因克隆及其表达分析

CLONING AND EXPRESSION ANALYSIS OF HEAT SHOCK PROTEIN 70 GENE FROM HYRIOPSIS CUMINGII

  • 摘要: 对三角帆蚌HSP70基因序列进行全长克隆及其分子生物学分析,并检测其在不同水温刺激下鳃组织中的表达变化。通过高通量转录组测序获得三角帆蚌HSP70基因(HcHSP70)长片段,采用3'RACE对其进行了3'末端克隆,经拼接得到HcHSP70 cDNA全长序列。采用多种分子生物学软件对HcHSP70 cDNA全长序列进行了特征分析,采用实时荧光定量PCR技术检测了其组织分布,并结合Western-blot技术检测蚌鳃中该基因mRNA与蛋白经不同水温刺激后的表达变化。结果显示,HcHSP70 cDNA全长为2298 bp,其中开放阅读框为1974 bp,编码657个氨基酸。预测分子量大小为71.6 Ku,pH7.0时的理论等电点为5.61。氨基酸序列分析表明,HcHSP70氨基酸序列含HSP70家族的3个标签序列(I9DLGTTYS16、I197FDLGGGTFDVSIL210和I336 VLVGGSTRIPKVQK350),与长牡蛎及泥蚶的HSP70同源性最高(91%)。实时荧光定量PCR检测结果显示,HcHSP70在鳃、性腺、肝胰腺、外套膜及肌肉等5种被检组织中均有表达,以肝胰腺中的表达水平最高。实时荧光定量PCR与Western-blot技术检测皆表明,蚌鳃组织中HcHSP70基因与蛋白的表达量在37℃时达到最高,而在40℃水温刺激下表达水平下调至正常值,表明其在适应高温刺激时发挥了重要作用。

     

    Abstract: In the present study, the cDNA sequence of Hyriopsis cumingii HSP70 (HcHSP70) was cloned by 3'rapid amplification of cDNA ends methods (3'-RACE) based on a long chain sequence of HcHSP70 and it was determined by high flux sequencing for transcriptome of H. cumingii blood cells, and its expression in the different tissues was detected with quantitative real time polymerase chain reaction (qRT-PCR). Results showed that the full-length of HcHSP70 cDNA was 2298 bp long including a 1974 bp open reading frame (ORF) encoding a polypeptide of 657 amino acids that estimated molecular mass of 71.6 Ku and an isoelectric point of 5.67. Sequence comparison indicated that HcHSP70 shares the highest identity (91%) with HSP70 of Tegillarca granosa and Crassostrea gigas. HcHSP70 mRNA expressed in all 5 detected tissues (hepatopancreas, gonad, gill, mantle, muscle) with the highest expression in hepatopancreas. Both mRNA and protein level of HcHSP70 in gill significantly up-regulated at 37℃ and rapidly reduced to normal level under 40℃.

     

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