草鱼nrf1基因的克隆与表达及其应激响应研究

CLONING,EXPRESSION AND STRESS RESPONSE OF NRF1 GENE IN GRASS CARP(CTENOPHARYNGODON IDELLA)

为研究跨膜转录因子核因子E2相关因子1(Nuclear factor-erythroid 2 related factor 1,nrf1)在动物机体抗氧化应激过程中的作用,通过同源克隆获得草鱼nrf1基因序列,其开放阅读框为1560 bp,编码519个氨基酸;系统进化树分析表明草鱼nrf1与团头鲂(Megalobrama amblycephala)进化关系较近。对nrf1进行组织表达分析,结果显示nrf1在肝脏中表达水平最高,其次是心脏和肠道(P<0.05)。昼夜节律分析显示,在9:00时nrf1表达水平最高且显著高于3:00、12:00、18:00、21:00和24:00(P<0.05)。经急性氨氮胁迫处理24h和48h时,发现草鱼nrf1基因在低氨氮(5 mg/L)组和高氨氮(20 mg/L)组中表达量相对于对照组均显著升高(P<0.05);且低氨氮组nrf1表达量在24h时显著低于高氨氮组(P<0.05),而在48h时显著高于高氨氮组(P<0.05)。此外,研究使用3种不同蛋白源(鱼粉、菜粕和豆粕)饲料对草鱼进行生长实验,发现在养殖后14d、28d和35d,菜粕和豆粕组nrf1表达水平显著高于鱼粉组(P<0.05),其中28d时豆粕组nrf1表达量显著高于菜粕组(P<0.05)。综上所述,草鱼nrf1基因表达具有组织特异性,且受到水体氨氮浓度和饲料蛋白源的调控,研究为揭示鱼类nrf1基因的分子特征及其抗氧化应激反应中的功能奠定了理论基础。

Nuclear erythroid 2 related factor 1(nrf1),a transmembrane transcription factor,plays an important role in anti-oxidative stress in animals.In this study,the nrf1 gene sequence of grass carp(Ctenopharyngodon idella)was obtained by homologous cloning,with an open reading frame(ORF)spanning 1560 bp which encoded a polypeptide consisting of 519 amino acids.Phylogenetic tree analysis showed that nrf1 was closely related to Megalobrama amblycephala.The tissue expression analysis of nrf1 revealed the liver as the primary site of nrf1 expression,followed by the heart and intestine.Circadian rhythm analysis showed that the expression level of nrf1 was the highest at 9:00 and significantly higher than that at 3:00,12:00,18:00,21:00 and 24:00(P<0.05).After 24h and 48h of acute ammonia stress treatment,the expression of nrf1 gene was significantly upregulated in both low ammonia nitrogen(5 mg/L)and high ammonia nitrogen(20 mg/L)groups compared to the control group(0)(P<0.05).The expression level of nrf1 was significantly lower in the low ammonia nitrogen group compared to the high ammonia nitrogen group at 24h(P<0.05),and significantly higher than that in the high ammonia nitrogen group at 48h(P<0.05).Furthermore,growth experiments on grass carp were conducted using three distinct protein sources(fish meal,rapeseed meal,and soybean meal),revealing that the expression level of nrf1 was significantly higher in the rapeseed meal and soybean meal groups compared to the fish meal group at 14d,28d,and 35d after feeding trial(P<0.05).At 28d,the expression level of nrf1 in the soybean meal group was notably higher than that in the rapeseed meal group.In conclusion,the expression of the nrf1 gene in grass carp exhibits tissue specificity and is regulated by ammonia nitrogen concentration in water as well as dietary protein source.This study provides a theoretical framework for elucidating the molecular characteristics of the nrf1 gene in fish and its pivotal role in mediating antioxidative stress response.