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伴侣蛋白CCT在蛋白折叠、抗胁迫以及调节细胞生长等生理过程中担负重要作用。研究利用同源克隆和RACE技术克隆了皱纹盘鲍伴侣蛋白CCTζ(HdhCCTζ)cDNA全长序列,并对CCTζ基因的序列特征进行了分析。HdhCCTζcDNA序列长1960 bp,开放阅读框为1599 bp,编码532个氨基酸,生物学软件推测其编码蛋白相对分子量为58.46 kD,等电点为6.53。BLAST分析结果表明HdhCCTζ与哺乳动物、鸟和鱼类等物种的CCT具有较高的同源性。生物信息学分析结果显示,HdhCCTζ蛋白序列中包含3个典型的CCT信号基序(35RTNLGPKGTLKML47,56LTKDGNVLLHEMQIQHP72和84QDDITGDGT92)以及1个腺苷三磷酸结合基序(89GDGTTS94)。此外,在HdhCCTζ蛋白序列中还包含3个糖基化位点(23NISA26,128NKSL131and234NVSL237)。实时荧光定量PCR检测结果显示,HdhCCTζ在皱纹盘鲍组织中为组成型表达,但主要集中在性腺、血淋巴和肝胰腺中表达。使用不同锌含量(6.69 mg/kg、33.85 mg/kg、710.63 mg/kg和3462.5 mg/kg)的日粮来饲喂幼鲍20周后,取其肝脏和血细胞总RNA,利用实时荧光定量PCR技术进行HdhCCTζmRNA的表达水平分析。分析结果显示,随着日粮中锌含量的增加,HdhCCTζmRNA在血淋巴和肝胰腺中的表达水平呈现上调的趋势。相比于锌适量组(33.85 mg/kg),过量的锌(3462.5 mg/kg)却能够下调HdhCCTζmRNA的表达水平。上述结果表明,HdhCCTζ的表达受到日粮中锌添加量的影响,而高表达量的HdhCCTζ可能在提高皱纹盘鲍机体抗胁迫过程中发挥重要作用。
The chaperone CCT plays an important role in physiological processes such as protein folding, resistance to stress and regulation of cell growth. The full-length cDNA sequence of CCTζ (HdhCCTζ) was cloned by homology cloning and RACE techniques. The sequence characteristics of CCTζ gene were also analyzed. The cDNA sequence of HdhCCTζ was 1960 bp in length and 1599 bp in open reading frame (ORF) encoding 532 amino acids. The biological software estimated the relative molecular mass of the encoded protein was 58.46 kD and the isoelectric point was 6.53. BLAST analysis showed that HdhCCTζ has high homology with CCT in mammals, birds and fish. Bioinformatics analysis showed that the HdhCCTζ protein sequence contained three typical CCT signal motifs (35RTNLGPKGTLKML47, 56LTKDGNVLLHEMQIQHP72 and 84QDDITGDGT92) and one adenosine triphosphate binding motif (89GDGTTS94). In addition, three glycosylation sites (23NISA26, 128NKSL131 and 234NVSL237) were also included in the HdhCCTζ protein sequence. Real-time quantitative PCR results showed that HdhCCTζ was constitutively expressed in abalone abalone but mainly in gonads, hemolymph and hepatopancreas. After 20 weeks of feeding, the total RNA of liver and blood cells was fed on diets containing different levels of zinc (6.69 mg / kg, 33.85 mg / kg, 710.63 mg / kg and 3462.5 mg / kg) Technique to analyze the expression level of HdhCCTζmRNA. The results showed that the expression of HdhCCTζmRNA in the hemolymph and hepatopancreas showed an upward trend as the dietary zinc content increased. Excess zinc (3462.5 mg / kg) was able to down-regulate the expression of HdhCCTζ mRNA compared to the zinc-equivalent group (33.85 mg / kg). The above results indicate that the expression of HdhCCTζ is affected by the amount of zinc added in the diet, whereas the high expression level of HdhCCTζ may play an important role in enhancing the body’s anti-stress abilities.