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

doi:10.3785/j.issn.1008-9209.2021.01.182

Journal of Zhejiang University (Agriculture and Life Sciences)

2021, Vol. 47

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

Animal sciences & veterinary medicine

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

Download:

HTML

HTML ( )

PDF(4587KB)
Export: BibTeX | EndNote (RIS)

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

Received: 18 January 2021 Published: 27 October 2021

CLC:

S 917.4

Corresponding Authors: Xilei LI E-mail: sushiping@ahau.edu.cn;xlli@ahau.edu.cn

	Service
	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	Shiping SU
	Qingqing LI
	Qiming XIE
	Fan LIU
	Jun ZHANG
	Xilei LI

Cite this article:

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.

URL:

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

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

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

Table 1 Primer sequences and function

Fig. 1 Nucleic acid and amino acid sequence analysis of NUCB2 in Myxocyprinus asiaticus5′ and 3′ untranslated regions (UTR) are indicated by italics letters; gray highlighted letters represent putative signal peptides; the propeptide cleavage sites for the predicted processing are indicated by boxes; the Nesfatin-1 (M30) peptide region is underlined in black, and the Nesfatin-2 peptide region is underlined with a dashed line, and the Nesfatin-3 peptide region is underlined with a wavy line; the predicted tail signal is circled with an ellipse; single asterisk (*) indicates a stop codon.

Fig. 2 Homology alignment of NUCB2 amino acid sequences in M. asiaticusThe highly conserved amino acid sequences are represented by the black shading, and the moderately conserved amino acid sequences and the non-conservative amino acid sequences are represented by the grey shading and the colorless shading, respectively. The signal peptide region is indicated by the red brackets; and the predicted cleavage sites are indicated by the red boxes, where the structural domains of Nesfatin-1, Nesfatin-2 and Nefatin-3 are successively divided.

Fig. 3 Phylogenetic tree based on multiple alignments of NUCB2 amino acid sequences from various species

Fig. 4 Relative expression levels of Nesfatin-1 mRNA in the diencephalon and hepatopancreas of two developmental stages of M. asiaticusThe same lowercase letter above the bars indicates no significant differences between different fish ages of the same tissue at the 0.05 probability level, and different uppercase letters above the bars indicate highly significant differences between different tissues of the same fish age at the 0.01 probability level.

Fig. 5 SDS-PAGE identification of prokaryotic expression protein Nesfatin-1 of M. asiaticus

Fig. 6 Distribution of Nesfatin-1 in diencephalon and hepatopancreas of M. asiaticusA. Hepatopancreas; B. Amplification view of the scribed areas of Pc in the Figure A; C. Diencephalon; D. Amplification view of the scribed areas of Na in the Figure C. HP: Hepatopancreas; Pc: Pancreatic cell; TV: The third ventricle; Nl: Lateral tuberal nucleus; Na: Anterior periventricular nucleus; Np: Preoptic nucleus magnocelluar part. White arrow shows the pancreatic cells; red arrow shows the magnocelluar neuron.


[1]	OH-I S, SHIMIZU H, SATOH T, et al. Identification of nesfatin-1 as a satiety molecule in the hypothalamus. Nature, 2006,443(7112):709-712. DOI:10.1038/nature05162 doi: 10.1038/nature05162

[2]	SCHALLA M A, UNNIAPPAN S, LAMBRECHT N W G, et al. NUCB2/nesfatin-1 inhibitory effects on food intake, body weight and metabolism. Peptides, 2020,128:170308. DOI:10.1016/j.peptides.2020.170308 doi: 10.1016/j.peptides.2020.170308

[3]	GONZALEZ R, KERBEL B, CHUM A, et al. Molecular, cellular and physiological evidences for the anorexigenic actions of Nesfatin-1 in goldfish. PLoS ONE, 2010,5(12):e15201. DOI:10.1371/journal.pone.0015201 doi: 10.1371/journal.pone.0015201

[4]	HATEF A, SHAJAN S, UNNIAPPAN S. Nutrient status modulates the expression of nesfatin-1 encoding nucleobindin 2A and 2B mRNAs in zebrafish gut, liver and brain. General and Comparative Endocrinology, 2015,215:51-60. DOI:10.1016/j.ygcen.2014.09.009 doi: 10.1016/j.ygcen.2014.09.009

[5]	ZHANG X, WANG S Y, CHEN H, et al. The inhibitory effect of NUCB2/nesfatin-1 on appetite regulation of Siberian sturgeon (Acipenser baerii Brandt). Hormones and Behavior, 2018,103:111-120. DOI:10.1016/j.yhbeh.2018.06.008 doi: 10.1016/j.yhbeh.2018.06.008

[6]	NAKATA M, GANTULGA D, SANTOSO P, et al. Paraventricular NUCB2/Nesfatin-1 supports oxytocin and vasopressin neurons to control feeding behavior and fluid balance in male mice. Endocrinology, 2016,157(6):2322-2332. DOI:10.1210/en.2015-2082 doi: 10.1210/en.2015-2082

[7]	WILZ A M, WERNECKE K, APPEL L, et al. Endogenous NUCB2/Nesfatin-1 regulates energy homeostasis under physiological conditions in male rats. Hormone and Metabolic Research, 2020,52(9):676-684. DOI:10.1055/a-1196-2059 doi: 10.1055/a-1196-2059

[8]	PSILOPANAGIOTI A, NIKOU S, PAPADAKI H. Nucleobindin-2/Nesfatin-1 in the human hypothalamus is reduced in obese subjects and colocalizes with oxytocin, vasopressin, melanin-concentrating hormone, and cocaine- and amphetamine-regulated transcript. Neuroendocrinology, 2019,108(3):190-200. DOI:10.1159/000496731 doi: 10.1159/000496731

[9]	MAEJIMA Y, KUMAMOTO K, TAKENOSHITA S, et al. Projections from a single NUCB2/nesfatin-1 neuron in the paraventricular nucleus to different brain regions involved in feeding. Brain Structure Function, 2016,221(9):4723-4731. DOI:10.1007/s00429-015-1150-4 doi: 10.1007/s00429-015-1150-4

[10]	BERTUCCī J I, BLANCO A M, SáNCHEZ-BRETA?O A, et al. Ghrelin and NUCB2/Nesfatin-1 co-localization with digestive enzymes in the intestine of pejerrey (Odontesthes bonariensis). The Anatomical Record, 2019,302(6):973-982. DOI:10.1002/ar.24012 doi: 10.1002/ar.24012

[11]	SUN S J, YANG H W. Tissue-specific localization NUCB2/nesfatin-1 in the liver and heart of mouse fetus. Development Reproduction, 2018,22(4):331-339. DOI:10.12717/dr.2018.22.4.331 doi: 10.12717/dr.2018.22.4.331

[12]	DORE R, LEVATA L, LEHNERT H, et al. Nesfatin-1: functions and physiology of a novel regulatory peptide. Journal of Endocrinology, 2017,232(1):R45-R65. DOI:10.1530/joe-16-0361 doi: 10

[13]	BLANCO A M, VELASCO C, BERTUCCI J I, et al. Nesfatin-1 regulates feeding, glucosensing and lipid metabolism in rainbow trout. Frontiers in Endocrinology, 2018,9:484-496. DOI: 10.3389/fendo.2018.00484 doi: 10.3389/fendo.2018.00484

[14]	GATTA C, DE FELICE E, D’ANGELO L, et al. The case study of Nesfatin-1 in the pancreas of Tursiops truncatus. Frontiers in Endocrinology, 2018,9:1845-1851. DOI:10.3389/fphys.2018.01845 doi: 10.3389/fphys.2018.01845

[15]	BERTUCCI J I, BLANCO A M, CANOSA L F, et al. Glucose, amino acids and fatty acids directly regulate ghrelin and NUCB2/nesfatin-1 in the intestine and hepatopancreas of goldfish (Carassius auratus) in vitro. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2017,206:24-35. DOI:10.1016/j.cbpa.2017.01.006 doi: 10.1016/j.cbpa.2017.01.006

[16]	XU L, WANG Q L, GUO F F, et al. Nesfatin-1 signaling in the basomedial amygdala modulates the gastric distension-sensitive neurons discharge and decreases gastric motility via melanocortin 3/4 receptors and modified by the arcuate nucleus. European Journal of Pharmacology, 2015,764:164-172. DOI:10.1016/j.ejphar.2015.07.002 doi: 10.1016/j.ejphar.2015.07.002

[17]	LIU D Q, ZHOU Y, YANG K, et al. Low genetic diversity in broodstocks of endangered Chinese sucker, Myxocyprinus asiaticus: implications for artificial propagation and conser-vation. Zookeys, 2018,792:117-132. DOI:10.3897/zookeys.792.23785 doi: 10.3897/zookeys.792.23785

[18]	YE J S, ZHAO S Z, YUAN S, et al. Study on resource conservation of Chinese sucker (Myxocyprinus asiaticus). Agricultural Biotechnology, 2019,8(1):97-100, 125. DOI:10.19759/j.cnki.2164-4993.2019.01.026 doi: 10

[19]	BERNIER N J, KRAAK G VAN DER, FARRELL A P, et al. Fish Neuroendocrinology. London, UK: Academic Press, 2009:12-20. DOI:10.1016/c2009-0-01701-2 doi: 10.1016/c2009-0-01701-2

[20]	SHIMIZU H, OH-I S, HASHIMOTO K, et al. Peripheral administration of Nesfatin-1 reduces food intake in mice: the leptin-independent mechanism. Endocrinology, 2009,150(2):662-671. DOI:10.1210/en.2008-0598 doi: 10.1210/en.2008-0598

[21]	RIVA M, NITERT M D, VOSS U, et al. Nesfatin-1 stimulates glucagon and insulin secretion and beta cell NUCB2 is reduced in human type 2 diabetic subjects. Cell and Tissue Research, 2011,346(3):393-405. DOI:10.1007/s00441-011-1268-5 doi: 10.1007/s00441-011-1268-5

[22]	NOZAWA S, KIMURA T, KURISHIMA M, et al. Analyses of a satiety factor NUCB2/nesfatin-1; gene expressions and modulation by different dietary components in dogs. Journal of Veterinary Medical Science, 2016,78(3):411-417. DOI:10.1292/jvms.15-0255 doi: 10

[23]	BANERJEE S, CHATURVEDI C M. Nesfatin-1: locali-zation and expression in avian gonads and its modulation by temporal phase relation of neural oscillations in female Japanese quail, Coturnix coturnix japonica. General and Comparative Endocrinology, 2015,224:205-215. DOI:10.1016/j.ygcen.2015.08.016 doi: 10.1016/j.ygcen.2015.08.016

[24]	YANG M L, ZHANG Z H, WANG C, et al. Nesfatin-1 action in the brain increases insulin sensitivity through Akt/AMPK/TORC2 pathway in diet-induced insulin resistance. Diabetes, 2012,61(8):1959-1968. DOI:10.2337/db11-1755 doi: 10.2337/db11-1755

[25]	GONZALEZ R, SHEPPERD E, THIRUPPUGAZH V, et al. Nesfatin-1 regulates the hypothalamo-pituitary-ovarian axis of fish. Biology of Reproduction, 2012,87(4):1-11. DOI:10.1095/biolreprod.112.099630 doi: 10.1095/biolreprod.112.099630

[26]	MOHAN H, RAMESH N, MORTAZAVI S, et al. Nutrients differentially regulate nucleobindin-2/nesfatin-1 in vitro in cultured stomach ghrelinoma (MGN3-1) cells and in vivo in male mice. PLoS ONE, 2014,9(12):e115102. DOI:10.1371/journal.pone.0115102 doi: 10.1371/journal.pone.0115102

[27]	STENGEL A, GOEBEL M, WANG L X, et al. Central nesfatin-1 reduces dark-phase food intake and gastric emptying in rats: differential role of corticotropin-releasing factor (2) receptor. Endocrinology, 2009,150(11):4911-4919. DOI:10.1210/en.2009-0578 doi: 10.1210/en.2009-0578

[28]	王庆,刘琼瑜,李益祺,等.Nesfatin-1调控斜带石斑鱼生殖作用的研究.渔业研究,2016,38(1):5-13. DOI:10.14012/j.cnki.fjsc.2016.01.002 WANG Q, LIU Q Y, LI Y Q, et al. The study of nesfatin-1 in Epinephelus coioides reproductive regulation. Journal of Fisheries Research, 2016,38(1):5-13. (in Chinese with English abstract) doi: 10.14012/j.cnki.fjsc.2016.01.002

[1]	Huichun LIU,Jiaqiang ZHANG,Guangying MA,Jianghua ZHOU,Wenting XU,Kaiyuan ZHU. *Cloning of PsDHN1* gene of Paeonia suffruticosa and waterlogging tolerance analysis of transgenic Arabidopsis with PsDHN1 gene**[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2021, 47(3): 335-346.

[2]	Hui LI,Wei FENG,Junjie YU,Mingyin ZHANG,Chunmiao ZHOU,Yongkai TANG. Research progress of peroxiredoxingene in crustaceans[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2021, 47(3): 284-294.

[3]	Xuedong ZHANG,Huanhuan WANG,Ying GE,Dandan SONG,Lei ZHANG,Qinghai LI,Lifeng LOU. Cloning and tissue expression of alternative spliceosome in chicken G-protein alpha subunit gene[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(5): 603-610.

[4]	XU Kangkang, DING Tianbo, YAN Yi, LI Can, YANG Wenjia. *Expression analysis of glutathione S-transferase genes in Lasioderma serricorne* (Coleoptera: Anobiidae) subjected to CO₂-enriched atmosphere**[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2017, 43(5): 599-607.

[5]	ZHENG Qunyan, PAN Xiaoyi, SHEN Jinyu, CHEN Shaobo, XU Yang, XU Ting. *Molecular cloning and tissue expression analysis of glutamate dehydrogenase gene from Macrobrachium rosenbergii* under MrTV infection stress**[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2017, 43(5): 639-648.

[6]	WANG Jiaqing, DONG Huiming, LI Zhengang, LI Shaoming, WANG Ruonan, FU Yujie. Cloning and function prediction of full-length cDNA for cathepsin E derived from medaka (Oryzias latipes).[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2017, 43(2): 183-191.

[7]	JIANG Ming, LIU Qing’e, ZHANG Yanru, ZHU Qi, GONG Xiu, YU Keke, ZHOU Xiuqian. Cloning and expression of a C3H-type zinc finger protein gene BoCCCH2 from Brassica oleracea var. italica.[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2016, 42(2): 143-149.

[8]	Jiang Ming, Chen Beibei, Guan Ming, Li Jinzhi, Huang Xiaomei, Gu Yunji . Cloning and expression analysis of a transcription factor gene BoWRKY2 from broccoli.[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2015, 41(2): 153-159.

[9]	XU Hongna, YU Hongwei. Cloning, expression of carboxylesterase BioH and improvement of its hydrolysis activity by directed evolution*[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2013, 39(6): 607-612.

[10]	LU Yongquan1, JIA Qing1, TONG Zaikang1， CHEN Jianyang2. Cloning and sequence analysis of three novel chalone synthase genes in Cryptomeria plants*[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2013, 39(3): 246-252.

[11]	. Cloning and identification of human colipaselike 3 and its homologous cDNAs[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2012, 38(5): 575-584.

[12]	ZHANG Yongliang, SHENG Dongfeng, ZHU Yong. Cloning and characterization of Bombyx mandarina prophenoloxidase gene PPO1[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2012, 38(3): 256-262.

[13]	SUN Yue na,XU Tian jun,SHI Ge,WANG Ri xin. Molecular cloning and expression analysis of stress inducible protein I gene of Miichthys miiuy.[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2012, 38(2): 159-165.

[14]	YU Chong, HUANG Yong, TIAN Ming-xing,SHI Min,LI Min,ZHAO Fang-fang. Cloning and expression of reticuloendotheliosis virus (REV) and development of indirectELISA for detection ofantibody to REV[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2011, 37(4): 363-370.

[15]	JIANG Ming,CHEN Xiao-shang,LI Jin-zhi. Cloning, expression and sequence analysis of anthocyanidin synthase gene BcANS in Brassica campestris var. purpurea[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2011, 37(4): 393-398.

Viewed

Full text

Abstract

Cited

Shared

Discussed