刀鲚PPARγ基因的cDNA克隆及其应激应答

Molecular cloning and stress response of PPARγ in Coilia nasus

旨在研究刀鲚(Coilia nasus)过氧化物酶体增殖物激活受体γ(peroxisome proliferator activated receptor, PPARγ)基因的应激调控表达,克隆并获得了刀鲚PPARγ基因的cDNA全长。刀鲚PPARγ基因的cDNA全长1951 bp,开放阅读框1470 bp,预测编码489个氨基酸。刀鲚PPARγ包括4个功能结构域,即A/B区, DNA结合区(DNA binding domain, DBD区),铰链区,配体结合区(ligand binding domain, LBD区)。运用荧光定量PCR (real time quantitative PCR, RT-qPCR)检测刀鲚PPARγ基因在不同组织、运输胁迫和胚胎发育时期的表达。结果显示,刀鲚PPARγ在各组织中均有表达,其中在肝中表达量最高,在脑、肠、心脏、肾、头肾、肌肉中相对高表达,在鳃和脾中微量表达。运输胁迫过程中, PPARγ基因表达显著上调(P<0.05),在4 h达到峰值,随后显著降低,但仍高于对照组。PPARγ在胚胎发育时期各时期均表达,其中在受精卵时期高表达,随后表达量急剧降低,并在此后的时期一直处于较低的表达水平。PPARγ基因在应激过程中发挥重要作用,也是胚胎发育过程中重要的基因。本研究为刀鲚的人工繁育和应激调控提供了理论基础。

Coilia nasus is a commercially important fish species that is valued for its nutritive value and is used as a delicacy. It is widely distributed in the Yangtze River, coastal waters of China and Korea, and Ariake Sound in Japan. C. nasus is highly responsive to stress, which often causes tissue damage, apoptosis, and ultimately death during loading and transport, hampering the development of this industry. Peroxisome proliferator-activated receptors(PPARs) are ligand-dependent transcription factors belonging to the nuclear receptor superfamily. Like other nuclear receptors, PPARs consist of four domains that have distinct functions. The A/B domain, located in conserved N-terminus, is responsible for ligand-independent transcription activity. The C domain, which is the DNA-binding domain(DBD), contains two zinc finger structures. The D domain, the hinge region, connects the DBD with the ligand-binding domain(LBD) and is involved in the conformational changes of PPARs. The E/F domain is an LBD. Transcriptional activation by PPARs requires the presence of PPAR response elements(PPREs)in the promoter of the target gene. PPARs bind PPREs as heterodimers with retinoid X receptor. Three isoforms of PPARs, PPARα, PPARβ, and PPARγ, have been cloned in various mammals and fish. PPARγ is involved in many processes such as ontogenesis, lipid metabolism regulation, peroxisome biogenesis, immune functions and inflammation, and oxidative stress. The aim of this study was to investigate the stress response and expression of PPARγ during the embryonic development of C. nasus. Full-length PPARγ cDNA was obtained, which contained1951 base pairs with an open reading frame of 1470 base pairs, encoding 489 amino acids. PPARγ has four functional domains, A/B region, DBD, hinge region, and LBD. Real-time quantitative PCR was used to detect the expression of PPARγ in different tissues and under transport stress. The results showed that C. nasus PPARγ was expressed in all tissues, with the highest expression observed in the liver, relatively high expression detected in the brain, intestine, heart, kidney, head kidney, and muscle, and low expression in the gill and spleen. During transport stress, PPARγ expression was significantly up-regulated(P<0.05), peaked at 4 h, and was then significantly down-regulated. High expression of PPARγ may increase lipid metabolism and relieve inflammation and apoptosis.PPARγ plays an important role in lipid metabolism and stress responses. During embryonic development, PPARγshows the highest expression in the fertilized egg stage, after which its expression is rapidly decreased and then remains at a lower level. Lower expression of PPARγ may down-regulate lipid metabolism and transportation. This study provides information regarding the theoretical basis of stress regulation.