鲫鱼(Carassius auratus)半胱亚磺酸脱酸酶CSAD基因克隆及其表达研究

本研究以鲫鱼(Carassius auratus)为研究对象,采用巢式PCR和RACE技术克隆鲫鱼半胱亚磺酸脱酸酶CSAD基因c DNA序列,并且利用实时荧光定量PCR检测了CSAD m RNA在鲫鱼不同组织和昼夜节律中的相对表达水平,同时还研究了牛磺酸对鲫鱼肠道CSAD m RNA表达的影响。结果表明:(1)鲫鱼CSAD基因c DNA序列包含186bp的5?UTR序列,675bp的3?UTR序列,1503bp开放阅读框,编码500个氨基酸。同源性分析表明,鲫鱼和鲤鱼的同源性为97.2%。系统发育分析表明,鲫鱼与鲤鱼之间的亲缘关系最接近,置信度为100。经预测,其编码的蛋白质的分子量和等电点分别为56.82k Da和5.77;(2)Ca CSAD m RNA在肌肉、心脏、肠道及肝脏中的表达水平较高,在脑和鳃组织中的相对表达量较低;(3)鲫鱼肠道CSAD m RNA的相对表达量在6:00am点时最高,9:00pm点时相对表达量最低;(4)鲫鱼CSAD的相对表达丰度随着牛磺酸添加量的增加而逐渐下降。本研究结果不仅有助于理解鲫鱼CSAD基因的分子特征,同时将为进一步研究鱼类CSAD营养调控功能提供理论依据。

MOLECULAR CLONING AND EXPRESSION OF CSAD IN CARASSIUS AURATUS

The CSAD (cysteine sulfinic acid decarboxylase) cDNA sequence in Carassius auratus was cloned using nested PCR (polymerase chain reaction) and RACE (rapid amplification of cDNA ends) techniques, and the relative expression levels of CSAD mRNA in different tissues and circadian rhythms fish and in the intestine of taurine-feed C. auratus were analyzed. The results show the following findings. (1) The cloned cDNA sequence of CSAD was 2364bp in length, including 186bp 5''UTR sequence, 675bp 3''UTR sequence and 1503bp open reading frame encoding 500 amino acid polypeptide. The homology analysis showed that CSAD gene from C. auratus had 97.2% uniformity with C. carpio. Phylogenetic analysis indicated that the C. auratus CSAD was most closely related to CSAD of C. carpio in confidence level of 100. The encoded protein molecular weight was predicted for 56.82kDa with a pI of 5.77. (2) The highest expression of the CSAD mRNA was observed in the muscle, heart, intestine, and liver, and the lowest in the brain and gill. (3) The highest expression of intestine CSAD mRNA was found at 6:00 am, and the lowest at 9:00 pm. (4) The relative abundance of the CSAD mRNA decreased gradually with the increase of taurine. Therefore, this work could deepen understanding on the molecular characteristics of CSAD in C. auratus, and provide a theoretical basis for future study on the nutritional regulation of fish CSAD genes.