中国明对虾MKK4基因克隆及其在氨氮胁迫下的表达分析

为初步研究中国明对虾MKK4的生物学功能,采用RACE技术克隆获得中国明对虾MKK4基因全长cDNA序列,并对该序列进行分析。结果显示,中国明对虾MKK4基因全长为2 064 bp,开放阅读框长1 221 bp,5'非编码区长214 bp,3'非翻译区长629 bp。将该基因命名为FcMKK4。推测该基因编码406个氨基酸,预测分子量为45.94 ku,理论等电点为8.50。同源性和系统进化分析发现FcMKK4与肩突硬蜱和印度跳蚁的同源性分别为80%和78%,与其他节肢动物MKK4聚为一支。荧光定量RT-PCR结果表明,FcMKK4基因在肌肉中的相对表达量最高,其次为肝胰腺。氨氮胁迫后该基因在中国明对虾肌肉、肝胰腺、血细胞、鳃、心脏、肠和胃中的表达量均显著增加,并有不同的时空表达谱式,表明FcMKK4可能参与中国明对虾非生物胁迫的应答反应。

cDNA cloning and expression analysis of MKK4 gene under ammonia-N stress in Fenneropenaeus chinensis

For preliminary research on the biology function of MKK4 in Fenneropenaeus chinensis in this study,full-length cDNA sequence of MKK4 gene in F.chinensis was cloned using RACE method and was named FcMKK4.The full-length of cDNA sequence of MKK4 is 2 064 bp,which contains a 214 bp 5'-UTR,629 bp 3'-UTR and 1 221 bp open reading frame(ORF)that encodes 406 amino acid residues.Its isoelectric point(PI)was 8.50 and molecular mass was 45.94 ku.Homology analysis revealed that the amino acid sequences of FcMKK4 were highly homogenous with MKK4 of other species,80% with Ixodes Scapularis,and 78% with Harpegnathos saltator.The phylogenetic analysis showed that F.chinensis FcMKK4 was in the same class with other arthropods' MKK4.The expression level of FcMKK4 gene in different tissues was analyzed by quantitative real-time PCR.The results indicated that the highest expression level of FcMKK4 gene was in muscle,and that in hepatopancreas was the second.Real-time PCR analysis showed that the expression level of FcMKK4 was up-regulated significantly in muscle,hepatopancreas,hemocytes,gill,heart,intestine and stomach after stimulation with ammonia-N stress,and FcMKK4 showed different expression profiles in different tissues.The results implied that FcMKK4 might play an important role in abiotic stress response in F.chinensis.