皱纹盘鲍伴侣蛋白CCTζ的分子克隆及其在不同锌含量日粮处理下的表达分析(英文)

MOLECULAR CLONING,CHARACTERIZATION AND EXPRESSION OF CHAPERONIN CONTAINING T-COMPLEX POLYPEPTIDE SUBUNIT (ZETA) TRANSCRIPT FROM ABALONE HALIOTIS DISCUS HANNAI INO IN RESPONSE TO DIETARY ZINC

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。

Chaperonin containing T-complex polypeptide （CCT） is believed to play crucial role in mediating protein folding and correlating with better cell growth in the physiological processes. In this study, the chaperonin containing T-complex polypeptide subunit （zeta） was cloned from abalone Haliotis discus hannai Ino （HdhCCTζ） by homology cloning with degenerate primers and RACE techniques. The full length of HdhCCT（cDNA was 1960 bp with a 1599 bp open reading frame （ORF） encoding 532 amino acids with a calculated molecular mass of 58.46 kD and a predicted isoelectric point of 6.53. BLAST analysis revealed that HdhCCTζ shared high identities with other known CCTs from mammal, bird and fish, etc. Sequence characterization revealed that HdhCCT~ contained three characteristic signature motifs （35RTNLGPKGTLKML47, 56LTKDGNVLLHEMQIQHP72 and 84QDDITGDGTgz）. And there was a putative adenosine triphosphate-binding domain （89GDGTTS94） in HdhCCTζ. In Addition, three potential N-glycosylation sites （23NISA26, 128NKSL131 and 234NVSL237） were also identified in HdhCCT~. Relatively higher levels mRNA expression were detected in gonad, haemocytes and hepatopancreas by real-time PCR assays. And the relative expression levels of HdhCCT（mRNA in hepatopancreas and haemocytes were detected by real-time PCR in abalone fed with four different diets containing graded levels of zinc （6.69, 33.85, 710.63 and 3462.5 mg/kg） for 20 weeks, respectively. The results showed that the expression levels of HdhCCT（ mRNA were higher at dietary zinc （33.85 mg/kg, 710.63 mg/kg and 3462.5 mg/kg） than these at zinc-deficient diets. But HdhCCT（mRNA expression levels were down-regulated by exces- sive concentration of dietary zinc （3462.5 mg/kg） compared with optimal dietary zinc （33.85 mg/kg）. It is implied that the expression levels of HdhCCTζ could be affected by dietary zinc in the hepatopancreas and haemocytes, and the rela- tive higher expression of HdhCCTζ was potentially involved in the anti-stress responses in Pacific abalone H. discus hannai.