拟穴青蟹MnSOD基因的克隆及表达分析

Molecular cloning and expression analysis of MnSODfrom mud crab Scylla paramamosain

超氧化物歧化酶(superoxide dismutase, SOD)可清除生物体内超氧阴离子自由基,有效地预防氧自由基对有机体的伤害。本研究从拟穴青蟹(Scylla paramamosain)的性腺中克隆了两种SOD基因的全长cDNA,分别为线粒体型锰型超氧化物歧化酶基因(SpmtMnSOD)和细胞质型锰型超氧化物歧化酶基因(SpcytMnSOD)。SpmtMnSOD基因cDNA全长1 154 bp,编码218个氨基酸,含有16个氨基酸的靶向线粒体的信号肽;SpcytMnSOD基因cDNA全长1 184 bp,编码286个氨基酸,未发现信号肽序列。两种MnSOD均含有锰型SOD的标签序列,以及4个保守的锰离子结合位点。荧光定量PCR分析显示SpmtMnSOD和SpcytMnSOD均主要在代谢活跃或参与免疫的心脏、鳃、卵巢和肝胰腺等器官中表达;均在卵巢O6期(完全成熟期)显著高于其他卵巢发育阶段,在精巢发育过程中表达量低,且没有显著性差异,推测其主要参与卵巢成熟过程自由基的清除。在副溶血弧菌(Vibrio parahemolyticus)感染后,SpmtMnSOD的表达量在鳃中(12 h)有所增加(P<0.05),SpcytMnSOD的表达量在肝胰腺(12 h)和鳃(3、12 h)中显著升高(P<0.05);在肽聚糖(peptidoglycan, PGN)刺激后,SpmtMnSOD的表达量在肝胰腺中(3、6、12 h)显著上升(P<0.05),SpcytMnSOD的表达量在肝胰腺(3、6、12 h)、鳃(6、24 h)和血(6、12 h)中均显著上调(P<0.05);在聚肌苷酸胞苷酸(polyinosinic:polycytidylic acid, Poly I:C)刺激后,SpmtMnSOD的表达量在肝胰腺(6、12 h)、鳃和血(3、6 h)中均显著增加(P<0.05),SpcytMnSOD的表达量在肝胰腺(12 h)、鳃(6、12 h)和血(3、6 h)中均显著增加(P<0.05)。推测这两种SOD参与了青蟹对外界环境的应激反应,对青蟹清除免疫应激产生的自由基起重要作用。

Superoxide dismutase(SOD)scavenges superoxide anion free radicals in organisms and effectively prevents the damage of oxygen free radicals to organisms.In the present study,the full-length cDNAs of two SOD genes:mitochondrial manganese superoxide dismutase(SpmtMnSOD)and cytoplasmic manganese superoxide dismutase(SpcytMnSOD),were cloned from the gonads of Scylla paramamosain.The full-length cDNA of SpmtMnSOD is 1154 bp,encoding a polypeptide of 218 amino acids(aa)that contains 16 aa signal peptides targeting mitochondria;the full-length cDNA of SpcytMnSOD is 1184 bp,which encodes a polypeptide of 286 amino acids without signal peptide sequence.Both two MnSODs contain the tag sequence of manganese-type SOD,as well as four conserved manganese ion binding sites.Quantitative real-time PCR was used to detect the expression of MnSODs gene in various tissues,gonadal development stages,and under different stresses.These results showed that the SpmtMnSOD and SpcytMnSOD were mainly expressed in the heart,gill,ovary and hepatopancreas,which are metabolically active or involved in the immune response.It is speculated that MnSODs play an important role in coping with oxidative stress and immune response.SpmtMnSOD and SpcytMnSOD were significantly higher in O6(tertiary vitellogenesis)than those of other ovarian developmental stages,and their expression levels were low during the testis development.It is also speculated that they are involved in the scavenging of free radicals during ovarian maturation.After infected by Vibrio parahemolyticus,compared to the control group,the expression of SpmtMnSOD increased in gills(12 h,P<0.05),and the expression of SpcytMnSOD significantly increased in hepatopancreas(12 h)and gills(3 h,12 h,P<0.05).After the challenge with peptidoglycan(PGN),compared to the control group,the expression of SpmtMnSOD was significantly increased in the hepatopancreas(3,6,12 h,P<0.05),and the expression of SpcytMnSOD was significantly increased in the hepatopancreas(3,6,12 h),gills(6,24 h)and haemocytes(6,12 h,P<0.05).After the challenge with polyinosinic:polycytidylic acid(Poly I:C),compared to the control group,the expression level of SpmtMnSOD in hepatopancreas(6,12 h),gills and haemocytes(3,6 h)and the expression level of SpcytMnSOD in hepatopancreas(12 h),gills(6,12 h)and haemocytes(3,6 h)were all up-regulated(P<0.05).It is thus speculated that they participate in the stress response to the external environment stimulus,balance the redox system in the body,and play an important role in removing free radicals generated by immune stress in mud crabs.