胭脂鱼核连蛋白2/Nesfatin-1基因克隆及其在间脑与肝胰脏中的差异表达

Cloning of nucleobindin-2/Nesfatin-1 gene and its differential expression in diencephalon and hepatopancreas of Chinese sucker(Myxocyprinus asiaticus)

为探索核连蛋白2(nucleobindin-2,NUCB2)对中国胭脂鱼(Myxocyprinus asiaticus)生长发育的调节规律,利用cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术克隆其cDNA全长,并应用实时荧光定量聚合酶链反应(quantitative real-time polymerase chain reaction,qPCR)和免疫荧光染色检测主要功能蛋白Nesfatin-1神经肽在3龄和6龄胭脂鱼间脑和肝胰脏中的表达分布。结果显示:NUCB2全长为2090 bp,包括99 bp的5′非翻译区、1449 bp的开放阅读框和542 bp的3′非翻译区,编码482个氨基酸,分为23个氨基酸编码的信号肽区和459个氨基酸编码的功能肽区,后者由前肽裂解位点赖氨酸106-精氨酸107和赖氨酸179-精氨酸180分成Nesfatin-1、Nesfatin-2和Nesfatin-33个功能区域,且Nesfatin-1的M30区域在鱼类中高度保守。在2个鱼龄期,Nesfatin-1在肝胰脏中的表达均极显著高于间脑(P<0.01),而在间脑和肝胰脏中的表达无明显龄期差异(P>0.05);在肝胰脏中其主要在米粒状的胰腺细胞中呈强阳性反应;而在间脑的结节外侧核、室周前核和室前核的少量大神经元细胞中呈阳性反应。综上所述,在胭脂鱼2个典型发育阶段,Nesfatin-1在间脑中的表达水平明显低于肝胰脏,说明Nesfatin-1对胭脂鱼生长发育的调节存在明显的中枢和外周差异。

In order to explore the regulation of nucleobindin-2(NUCB2)/Nesfatin-1 in the growth and development of Chinese sucker(Myxocyprinus asiaticu),the full-length cDNA sequence of NUCB2 was cloned by rapid amplification of cDNA ends(RACE)technology,and the expression and distribution of Nesfatin-1 in the diencephalon and hepatopancreas were observed by quantitative real-time polymerase chain reaction(qPCR)and immunofluorescence technique,respectively.The results showed that the full-length cDNA of NUCB2 was 2090 bp,including a 99 bp 5′untranslated region(UTR),a 1449 bp open reading frame(ORF)and a 542 bp 3′untranslated region.The NUCB2 gene encoded 482 amino acids,consisted of a signal peptide region of 23 amino acids and a NUCB2 peptide region of 459 amino acids.Two propeptide cleavage sites were in NUCB2 peptide region,lysine(Lys)106-arginine(Arg)107 and Lys179-Arg180,then three functional peptides of Nesfatin-1,Nesfatin-2,and Nesfatin-3 were divided according to the two cleavage sites.Phylogenetic tree analysis showed that the sequence of M30,the domain region of Nesfatin-1,was highly conserved in fish.In the same age,the expression level of Nesfatin-1 mRNA in the hepatopancreas was highly significantly higher than that in the diencephalon(P<0.01);however,in the same tissue,such as diencephalon and hepatopancreas,no significant differences were found between three and six-year-old M.asiaticus(P>0.05).The strong positive immunofluorescence signal was detected in the pancreatic cells instead of hepatocytes in the hepatopancreas.In the diencephalon,positive immunofluorescence signals were only detected in the lateral tuberal nucleus,anterior periventricular nucleus and preoptic nucleus magnocelluar part.In conclusion,the expression of Nesfatin-1 in the diencephalon is significantly lower than that in the hepatopancreas during two typical growth stages of Chinese sucker,indicating that the central and peripheral regulation of Nesfatin-1 on the growth and development of Chinese sucker has obvious difference.