马氏珠母贝热休克蛋白HSP60基因的克隆与表达分析

Cloning and Express Characters of HSP60 gene from Pinctada martensii

运用cDNA末端快速扩增(RACE)技术,克隆马氏珠母贝(Pinctada martensii)热休克蛋白HSP60基因cDNA全序列。序列全长2495 bp,开放阅读框(ORF)1734 bp,编码577个氨基酸,预测的分子量约为61.79 ku,理论等电点为5.49。5'非翻译区(5'UTR)长150 bp,3'非翻译区(3'UTR)长611 bp。同源性比对分析结果,马氏珠母贝HSP60基因与与太平洋长牡蛎(Crassostrea gigas)和光滑双脐螺(Biomphalaria glabrata)的同源性较高,达到75%。氨基酸序列分析显示,马氏珠母贝HSP60氨基酸序列具有典型的mt-HSP60特征序列、C-末端典型的GGM重复基序和一个ATP结合结构域。荧光定量数据分析发现,该基因在闭壳肌、外套膜、血淋巴、肝胰腺、性腺、鳃等6个组织中具有表达,在肝胰腺中表达量最高,鳃中次之,而在血液和闭壳肌中仅有少量表达;在脂多糖刺激后,该基因表达水平上调,12 h后达到最大值,之后又逐渐下调,均高于对照组,差异具统计学意义(P<0.05)。

To clarify the role ofPinctada martensii HSP60 in the immune and resilience, its full length cDNA sequence has been cloned. The full length of liSP60 is 2495 bp, including 150 bp of 5＇UTR, 611 bp of 3＇UTR, and 1734 bp of open reading frame. The open reading frame encodes a protein of 577 amino acids with an estimated molecular mass of 61.79 kDa and theoretical isoelectric point of 5.49. The alignment result show-s that the homology of the nuclear acid sequence of HSP60 from Rmartensii with that of the Crassostrea^oigas, Biomphalaria glabrata and other species was up to 75%. Analysis of the HSP60 amino acid sequence of Rmartensii show-s that it has a typical characteristic sequence of mt-HSP60, C-terminal typical repeat motifs GGM and an ATP binding domain. Results of qRT-PCR analysis show- that the P. martensii HSP60 was ubiquitously expressed in adductor muscle, mantle, hemolymph, hepatopancreas, gonads and gills, with the highest expression level in hepatopancreas and the second highest level in gill. The HSP60 expression level was extremely low in hemolymph and adductor muscle. Statistical analysis indicates that upon the LPS stimulation, the transcription level of HSP60 mRNA began to increase and reached the highest level after 12 h, and then gradually declined to the normal level （P〈0.05）.