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

doi:10.3785/j.issn.1008-9209.2021.01.182

浙江大学学报（农业与生命科学版）

2021, Vol. 47

Issue (5): 637-646 DOI: 10.3785/j.issn.1008-9209.2021.01.182

动物科学与动物医学

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

苏时萍(

),李卿青,谢启明,刘帆,张君,李西雷(

)

安徽农业大学动物科技学院，合肥 230036

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

Shiping SU(

),Qingqing LI,Qiming XIE,Fan LIU,Jun ZHANG,Xilei LI(

)

College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China

全文: PDF(4587 KB) HTML

摘要：

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2 090 bp，包括99 bp的5′非翻译区、1 449 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-3 3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。

关键词： 中国胭脂鱼; 核连蛋白2; 神经肽Nesfatin-1; 克隆; 差异表达

Abstract:

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 2 090 bp, including a 99 bp 5′ untranslated region (UTR), a 1 449 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.

Key words: Chinese sucker (Myxocyprinus asiaticus) nucleobindin-2 Nesfatin-1 cloning differential expression

收稿日期: 2021-01-18 出版日期: 2021-10-27

CLC:

S 917.4

基金资助: 农业农村部物种品种资源保护项目（渔业）(kj20180187);安徽省自然科学基金(1508085MC54);安徽省水产产业体系项目（〔2021〕711）

通讯作者: 李西雷 E-mail: sushiping@ahau.edu.cn;xlli@ahau.edu.cn

作者简介: 苏时萍（https://orcid.org/0000-0002-6380-372X），E-mail：sushiping@ahau.edu.cn

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引用本文:

苏时萍,李卿青,谢启明,刘帆,张君,李西雷. 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[J]. 浙江大学学报（农业与生命科学版）, 2021, 47(5): 637-646.

Shiping SU,Qingqing LI,Qiming XIE,Fan LIU,Jun ZHANG,Xilei LI. Cloning of nucleobindin-2/Nesfatin-1 gene and its differential expression in diencephalon and hepatopancreas of Chinese sucker(Myxocyprinus asiaticus). Journal of Zhejiang University (Agriculture and Life Sciences), 2021, 47(5): 637-646.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2021.01.182 或 http://www.zjujournals.com/agr/CN/Y2021/V47/I5/637

表1 引物序列及作用

图1 中国胭脂鱼NUCB2的核苷酸和氨基酸序列分析斜体字符区为5′和3′未翻译区；灰色突出显示的字母为假定信号肽；方框处为预测加工所需的前肽裂解位点；黑色下划线处为Nesfatin-1肽区（M30），虚线下划线处为Nesfatin-2肽区，波浪下划线处为Nesfatin-3肽区；用椭圆圈出的字符为预测的加尾信号；*表示终止密码子。

图2 中国胭脂鱼NUCB2氨基酸序列同源性比对黑色背景为高度保守的氨基酸序列，灰色背景为中度保守的氨基酸序列，无色背景为不保守的氨基酸序列；红色括号处为信号肽区域；红色方框处为预测的裂解位点，依次分割出Nesfatin-1、Nesfatin-2和Nefatin-3结构域。

图3 基于不同物种NUCB2氨基酸序列构建的系统发育树

图4 Nesfatin-1 mRNA在2个发育期胭脂鱼间脑和肝胰脏中的相对表达水平短栅上相同小写字母表示不同鱼龄的同一组织间在P＜0.05水平差异无统计学意义，不同大写字母表示相同鱼龄的不同组织间在P＜0.01水平差异有高度统计学意义。

图5 胭脂鱼Nesfatin-1原核表达蛋白的SDS-PAGE鉴定

图6 Nesfatin-1在中国胭脂鱼间脑及肝胰脏中的分布A.肝胰脏；B.图A中Pc划线区域的放大图；C.间脑；D.图C中Na划线区域的放大图。HP：肝胰脏；Pc：胰腺细胞；TV：第三脑室；Nl：结节外侧核；Na：室周前核；Np：室前核大细胞部。白色箭头表示米粒状胰腺细胞，红色箭头表示大神经元。

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