中华绒螯蟹Na^+-K^+-Cl^-协同转运蛋白基因的克隆和功能分析

Gene Cloning and Functional Analysis of Na^(+)-K^(+)-Cl^(-)Cotransporter from Eriocheir sinensis

为研究Na+-K+-Cl-协同转运蛋白(NKCC)基因在中华绒螯蟹(Eriocheir sinensis)应对高盐胁迫过程中的作用,采用RACE技术首次克隆到中华绒螯蟹NKCC基因全长序列,运用实时荧光定量PCR(qRT-PCR)对中华绒螯蟹NKCC基因进行组织定量,并进行生物学信息分析.结果表明:该基因的cDNA全长为4127bp,其中5'非编码区(UTR)长度为18bp,3'非编码区(UTR)长度为944bp,开放阅读框(ORF)长度为3165bp,编码1054个氨基酸.理化分析预测其分子量为51.066kDa,理论等电点为4.65.预测中华绒螯蟹NKCC二级结构由12个跨膜结构域组成.同源对比结果显示:中华绒螯蟹NKCC的氨基酸序列与拟穴青蟹(Scylla paramamosain)NKCC相似度最高,为86.17%.系统进化分析表明中华绒螯蟹NKCC与拟穴青蟹、三疣梭子蟹(Portunus trituberculatus)、可口美青蟹(Callinectes sapidus)、蓝蟹(Callinectes sapidus)聚为一支.不同组织的荧光定量结果显示:NKCC基因在中华绒螯蟹的肠、脑神经节、胸神经节、鳃、胃、肝胰腺、肌肉、眼、心脏和血清中均有表达且在中华绒螯蟹肠的表达量最高.在盐度为25‰的急性胁迫下,NKCC基因在中华绒螯蟹肠中的表达量随着胁迫时间延长变化显著(P<0.05),并在胁迫72h后恢复稳定.以上结果显示,NKCC基因参与了中华绒螯蟹渗透压调节,并在其渗透压平衡中发挥重要作用.

In order to study the role of Na^(+)-K^(+)-Cl^(-)cotransporter(NKCC)gene in the response of Chinese Mitten Crab(Eriocheir sinensis)to high salt stress,the full-length sequence of NKCC gene of Eriocheir sinensis was cloned for the first time by rapid amplification of cDNA ends(RACE).Quantitative analysis of NKCC gene in Eriocheir sinensis was carried out by qRT-PCR.The results showed that the full length of the cDNA was 4127 bp,including 18bp of UTR,944bp of UTR and 3165 bp of ORF,encoding 1054 amino acids.Physicochemical analysis predicted that the molecular weight was 51.066kDa and the theoretical isoelectric point was 4.65.It is predicted that the secondary structure of NKCC consists of 12 transmembrane domains.The results of homology comparison showed that the amino acid sequence of Eriocheir sinensis NKCC was 86.17%similar to that of Scylla paramamosain NKCC.Phylogenetic analysis showed that Chinese mitten crab NKCC was clustered into one clade with Scylla paramamosain,Portunus trituberculatus,Callinectes sapidus and Callinectes sapidus.The results of fluorescence quantitative analysis of different tissues showed that NKCC gene was expressed in intestine,brain ganglion,thoracic ganglion,gill,stomach,hepatopancreas,muscle,eye,heart and serum,and the highest expression level was found in intestine of Eriocheir sinensis.Under the acute stress of salinity of 25‰,the expression of NKCC gene in the intestine of Eriocheir sinensis changed significantly with the prolongation of stress time(P<0.05),and returned to stability after 72 hours of stress.These results indicate that NKCC gene is involved in osmotic pressure regulation and plays an important role in osmotic pressure balance of Eriocheir sinensis.