罗非鱼微囊藻毒素去毒相关基因克隆与活体表达研究

MOLECULAR CLONING AND IN VIVO EXPRESSION ANALYSIS OF MICROCYSTIN DETOXIFICATION-RELATED GENES IN NILE TILAPIA(OREOCHROMIS NILOTICUS)

可溶性谷胱甘肽S-转移酶(Soluble glutathione S-transferase,sGST)催化微囊藻毒素(Microcystins,MCs)与还原型谷胱甘肽(GSH)的加合去毒代谢过程,谷胱甘肽过氧化物酶(Glutathione peroxidase,GPX)为sGST的去毒反应提供GSH,解偶联蛋白2(Uncoupling protein 2,UCP2)则可抑制微囊藻毒素诱发活性氧导致的肝细胞凋亡。本研究从罗非鱼肝脏通过简并引物克隆sGST、GPX与UCP2基因cDNA核心序列,并应用5’RACE和3’RACE技术分别扩增罗非鱼肝脏sGST基因cDNA序列5’末端和3’末端序列而获得其cDNA全序列。罗非鱼肝脏sGST基因cDNA全序列长861 bp,其中5’非翻译区(5-’UTR)为25 bp,3’非翻译区(3-’UTR)为167 bp,开放阅读框(ORF)为669 bp,编码222个氨基酸,包含脊椎动物完整sGST的2个功能域:N-末端功能域(GSH结合位点)和C-末端功能域(底物结合位点)。罗非鱼sGST与真鲷、条石鲷(Oplegnathus fasciatus)、斑马鱼同源性较高,达到64.3%—78.5%,而与人、大鼠、小鼠、牛、猪、鸡差异较大,氨基酸同源性为48.2%—55.9%。罗非鱼肝脏GPX、UCP2基因cDNA核心序列长280 bp、776 bp,分别编码92、258个氨基酸。罗非鱼GPX与条石鲷、虹鳟、斑马鱼、人、大鼠、小鼠、牛、猪GPX同源性均较高,达到69.6%—85.9%。罗非鱼UCP2与真鲷、斑马鱼、鲤鱼、欧洲白鲑(Leuciscus cephalus)、草鱼、人、大鼠、小鼠UCP2同源性更高,达到71.8%—93.8%。通过对罗非鱼(5—8 g)活体腹腔注射亚致死量MC-LR(50μg/kg bwt),发现微囊藻毒素对罗非鱼肝脏sGST基因表达有显著的诱导作用(p<0.05),注射微囊藻毒素24h后sGST基因mRNA表达水平上调80%。注射微囊藻毒素24h后,虽然罗非鱼肝脏GPX与UCP2基因mRNA表达水平亦出现明显的升高趋势,但两者均未出现显著性的变化(p>0.05)。本研究从基因表达调控的角度证实,罗非鱼肝脏sGST在微囊藻毒素去毒过程中可能发挥关键作用,同时也说明罗非鱼肝脏GPX、UCP2基因可能在微囊藻去毒过程中发挥协同作用。

Soluble glutathione S-transferase（sGST） of freshwater fish is extremely important to microcystins（MCs） purification from fish body,by catalyzing the conjugation of GSH（Glutathione） with microcystins.Glutathione peroxidase（GPX） is essential to the detoxification of cyanotoxins by providing GSH for sGST.Because oxidation and reactive oxygen species（ROS） generation is necessarily involved in both the toxication and detoxification process of cyanotoxins in hepatocytes,uncoupling protein 2（UCP2） also has an important role in inhibiting the excessive production of ROS to restrain hepatocytes apoptosis.In this study,RT-PCR using degenerated primers,yielded a sGST cDNA fragment of 399 bp,a GPX cDNA fragment of 280 bp and a UCP2 cDNA fragment of 776 bp from the liver of a phytoplanktivorous freshwater fish,nile tilapia（Oreochromis niloticus）,which consumed substantial amounts of toxic blue-green algae in the food.The sGST cDNA fragment was further completed by 5＇ and 3＇ RACE（Rapid amplification of cDNA ends）.The full-length tilapia sGST cDNA was 861 bp in length,containing an ORF（Open reading frame） of 669 bp（encoding 222 amino acids）,flanked by 25 bp 5＇UTR（Untranslated region） and 167 bp 3＇UTR.The deduced amino acid sequence from this sGST cDNA fragment contains two conserved domains,N-terminal domain（glutathione-bindind site） and C-terminal domain（substrate-binding site）.Homology of the sGST amino acid sequence is high（64.3%—78.5%） with red sea bream（Pagrus major）,rock bream（Oplegnathus fasciatus） and zebrafish sGST,and is low（51.8%—55.9%） with human,rat,cow,pig and chicken sGST.However,both the GPX and UCP2 amino acid sequences show a high conservation with GPX and UCP2 of both fish and mammals（69.6%—85.9% for the GPX with rainbow trout,rock bream,zebrafish,human,rat,mouse,cow and pig GPX,and 71.8%—93.8% for the UCP2 with red sea bream,zebrafish,common carp,European chub Leuciscus cephalus,grass carp,human,rat and mouse UCP2）.Tilapia juveniles（5—8 g） were exposed to a sub-lethal dose（50μg/kg bwt） of MC-LR by intraperitoneal injection.Using β-actin as external control,a significant increase（about 80%） in the liver sGST mRNA expression was found in response to the MC-LR exposure after 24h（p〈 0.05）,indicating the importance of GST in microcystin detoxification.Although no significant changes were seen in the liver GPX and UCP2 mRNA expression,the expression level of both genes tended to increase after exposed to MC-LR.We suggested that sGST might be responsible for the strong tolerance of the phytoplanktivorous fish to microcystins,and hepatocyte proteins coping with oxidative stress（GPX and UCP2）,might also have some auxiliary effect.