闽西南黑兔<i>NPFFR</i>2基因的克隆与序列分析

桑雷; 孙世坤; 陈冬金; 王锦祥; 高承芳; 陈岩锋; 谢喜平

doi:10.19303/j.issn.1008-0384.2021.11.012

闽西南黑兔NPFFR2基因的克隆与序列分析

doi: 10.19303/j.issn.1008-0384.2021.11.012

福建省农业科学院畜牧兽医研究所，福建　福州　350013

基金项目: 国家兔产业体系福州综合试验站项目（CARS-43-G-5）；福建省科技计划公益类专项（2019R1026–16）

详细信息

作者简介:
桑雷（1981−），男，博士，研究方向：家畜遗传育种与繁殖（E-mail: sanglei1981@163.com）

通讯作者:
谢喜平（1966−），男，研究员，主要从事草食动物遗传育种与饲养（E-mail: xxp702@163.com）

中图分类号: S 811.6
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出版历程
- 收稿日期: 2021-08-05
- 修回日期: 2021-09-13
- 网络出版日期: 2021-12-30
- 刊出日期: 2021-11-28

Cloning and Bioinformatics of NPPFR2 of Minxinan Black Rabbit

Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350013, China

摘要

摘要: 目的神经肽FF受体2（Neuropeptide FF receptor 2，NPFFR2）是机体重要的神经内分泌因子神经肽FF（Neuropeptide FF，NPFF）的受体，参与糖皮质激素和焦虑行为的调节。测序分析闽西南黑兔NPFFR2基因，为其作为闽西南黑兔抗应激研究的重要靶基因提供数据。方法克隆NPFFR2基因，并对其进行测序及序列分析。结果成功克隆得到闽西南黑兔NPFFR2基因2个转录异构体，分别含1 815和1 824个核苷酸。它们拥有相同的5′-UTR（Untranslated region，非翻译区）和编码区序列（Coding region），但是在3′-UTR存在多个突变。根据mRNA二级结构预测结果，3′-UTR的突变致使NPFFR2基因转录异构体的二级结构存在部分差异。通过对兔NPFFR2基因进行遗传进化分析，发现其和人类的相关基因进化关系最近。依据编码序列对NPFFR2蛋白结构和亚细胞分布进行预测，NPFFR2蛋白含414个氨基酸，有7个跨膜α螺旋，是G蛋白偶联受体家族一员。结论分离克隆了闽西南黑兔NPFFR2基因的2种转录异构体，并利用生物信息学方法对其转录本和翻译蛋白的结构和功能进行了预测，为进一步高效利用闽西南黑兔NPFFR2基因提供相关理论支持。
- 闽西南黑兔 /
- NPFFR2 /
- 基因克隆
Abstract: Objective The gene related to an important neuroendocrine receptor of NPFF (neuropeptide FF), NPFFR2 (Neuropeptide FF receptor 2), that participates in the regulation of corticotropin-releasing hormone (CRT) and anxiety-like behaviors was analyzed for study on the stress response of Minxinan black rabbit. Method NPFFR2 was cloned, sequenced, and analyzed. Result Two transcript variants of NPFFR2 from Minxinan black rabbits were cloned to show the contents of 1 815 bp and 1 824 bp nucleic acid sequences. They had same sequences in 5′-UTR and coding region but multiple mutations in 3′-UTR. The predicted mRNA secondary structure displayed multiple mutations in the 3′-UTR which could cause changes on the structure as well as the function of NPFFR2. The phylogenetic analysis indicated the NPFFR2 from the rabbit was most closely related to that from Homo sapiens. According to the predicted protein structure and subcellular location, NPFFR2 protein belonged to the G protein-coupled receptors family and had 414 amino acids and 7 transmembrane α-helixes. Conclusion Two transcript variants of NPFFR2 from Minxinan black rabbits were cloned. The structures and functions of the transcripts and translated proteins were predicted by bioinformatics obtained. The results would be helpful for further study on the functions of NPFFR2 of Minxinan black rabbit.
- Minxinan black rabbit /
- NPFFR2 /
- cloning

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图 1 闽西南黑兔NPFFR2基因电泳结果

注：M，分子量标准；1为扩增条带。A，中间片段扩增；B，5’-RACE扩增；C，3’-RACE扩增；D，验证片段扩增。

Figure 1. PCR amplification of NPFFR2

Note: M: DNA marker; 1: amplified line; A: intermediate fragment PCR; B: 5’-RACE fragment PCR; C: 3’-RACE fragment PCR; D: verification fragment PCR.

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图 2 闽西南黑兔NPFFR2基因cDNA序列3′-UTR的序列比对

注：方框标识异构转录体间碱基序列差异。

Figure 2. Alignment of 3′-UTR cDNA sequence of NPFFR2 from Minxinan black rabbit

Note：Different nucleotides on transcript variants are boxed.

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图 3 NPFFR2基因mRNA二级结构预测分析

Figure 3. Predicted secondary mRNA structure of NPFFR2

下载: 全尺寸图片幻灯片

图 4 闽西南黑兔NPFFR2蛋白的结构和亚细胞定位预测

注：A，二级结构；B，三级结构；C，跨膜螺旋；D，亚细胞定位。箭头指向细胞膜。

Figure 4. Predicted structure and subcellular localization of NPFFR2 of Minxinan black rabbit

Note: A: Secondary structure; B: tertiary structure; C: transmembrane helices; D: subcellular localization. Arrow points at cell membrane.

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图 5 闽西南黑兔NPFFR2基因与其他物种的系统进化分析

Figure 5. Phylogenetic trees of NPFFR2 of Minxinan black rabbit and other animal species

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表 1 本研究使用的引物

Table 1. Primers applied

引物 Primers	序列（5′-3′） Sequence (5′-3′)	扩增长度 Length/bp	用途 Purpose
NPFFR2F1	GGCGTCACATCTGGACTGTC	1 322	NPFFR2 基因cDNA序列中间序列第一轮PCR扩增
NPFFR2R1	GGCTCATTTGACTCATGCAC		NPFFR2 基因cDNA序列中间序列第一轮PCR扩增
NPFFR2F2	CACCAGCCTCAAGTGGCAGC		NPFFR2 基因cDNA序列中间序列第二轮PCR扩增
NPFFR2R2	GAGTCACTGCGTAATACTGG		NPFFR2 基因cDNA序列中间序列第二轮PCR扩增
NPFFR2F3	CCAAGGAAGCTTCTGCCCTGAGAG	546	NPFFR2 基因cDNA序列3′-RACE扩增
NPFFR2F4	CAAGCAGGGACTGATGGGAGAAT	546	NPFFR2 基因cDNA序列3′-RACE扩增
NPFFR2R3	CAGTCTTCCCGGCACCAGTAGACA	564	NPFFR2 基因cDNA序列5′-RACE扩增
NPFFR2R4	ACAAATGCCGTCTTGATGGTGATC	564	NPFFR2 基因cDNA序列5′-RACE扩增
NPFFR2F5	TGCAGGTTCACACGTGGTCG	1 757	NPFFR2 基因cDNA序列验证
NPFFR2R5	AAGCAGACAACTCTATAGCC	1 757	NPFFR2 基因cDNA序列验证

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参考文献(24)

[1]	孙世坤, 桑雷, 陈冬金, 等. 闽西南黑兔的肉质分析研究 [J]. 中国农学通报, 2012, 28(20):33−36. doi: 10.11924/j.issn.1000-6850.2012-0630 SUN S K, SANG L, CHEN D J, et al. Analysis of minxinan black rabbit's meat quality [J]. Chinese Agricultural Science Bulletin, 2012, 28(20): 33−36.(in Chinese) doi: 10.11924/j.issn.1000-6850.2012-0630
[2]	COHEN S, JANICKI-DEVERTS D, MILLER G E. Psychological stress and disease [J]. JAMA, 2007, 298(14): 1685−1687. doi: 10.1001/jama.298.14.1685
[3]	ULRICH-LAI Y M, HERMAN J P. Neural regulation of endocrine and autonomic stress responses [J]. Nature Reviews Neuroscience, 2009, 10(6): 397−409. doi: 10.1038/nrn2647
[4]	BAO A M, SWAAB D F. The human hypothalamus in mood disorders: The HPA axis in the center [J]. IBRO Reports, 2019, 6: 45−53. doi: 10.1016/j.ibror.2018.11.008
[5]	AYACHI S, SIMONIN F. Involvement of mammalian RF-amide peptides and their receptors in the modulation of nociception in rodents [J]. Frontiers in Endocrinology, 2014, 5: 158.
[6]	GONCHARUK V, ZENG Z Z, WANG R P, et al. Distribution of the neuropeptide FF₁ receptor (hFF₁) in the human hypothalamus and surrounding basal forebrain structures: Immunohistochemical study [J]. The Journal of Comparative Neurology, 2004, 474(4): 487−503. doi: 10.1002/cne.20132
[7]	WU C H, TAO P L, HUANG E Y K. Distribution of neuropeptide FF (NPFF) receptors in correlation with morphine-induced reward in the rat brain [J]. Peptides, 2010, 31(7): 1374−1382. doi: 10.1016/j.peptides.2010.03.036
[8]	MANKUS J V, MCCURDY C R. Nonpeptide ligands of neuropeptide FF: Current status and structural insights [J]. Future Medicinal Chemistry, 2012, 4(9): 1085−1092. doi: 10.4155/fmc.12.67
[9]	孙瑜隆, 马小莉, 李迪杰, 等. 神经肽FF的生物活性研究进展 [J]. 现代生物医学进展, 2015, 15(7):1365−1368. SUN Y L, MA X L, LI D J, et al. Advances in the biological activity of neuropeptide FF [J]. Progress in Modern Biomedicine, 2015, 15(7): 1365−1368.(in Chinese)
[10]	JHAMANDAS J H, GONCHARUK V. Role of neuropeptide FF in central cardiovascular and neuroendocrine regulation [J]. Frontiers in Endocrinology, 2013, 4: 8.
[11]	ZAJAC J M. Neuropeptide FF: New molecular insights [J]. Trends in Pharmacological Sciences, 2001, 22(2): 63.
[12]	CONSTANTIN S, PIZANO K, MATSON K, et al. An inhibitory circuit from brainstem to GnRH neurons in male mice: A new role for the RFRP receptor [J]. Endocrinology, 2021, 162(5): bqab030. doi: 10.1210/endocr/bqab030
[13]	WACŁAWCZYK D, SILBERRING J, GRASSO G. The insulin-degrading enzyme as a link between insulin and neuropeptides metabolism [J]. Journal of Enzyme Inhibition and Medicinal Chemistry, 2021, 36(1): 183−187. doi: 10.1080/14756366.2020.1850712
[14]	ELSHOURBAGY N A, AMES R S, FITZGERALD L R, et al. Receptor for the pain modulatory neuropeptides FF and AF is an orphan G protein-coupled receptor [J]. Journal of Biological Chemistry, 2000, 275(34): 25965−25971. doi: 10.1074/jbc.M004515200
[15]	MOLLEREAU C, MAZARGUIL H, MARCUS D, et al. Pharmacological characterization of human NPFF₁ and NPFF₂ receptors expressed in CHO cells by using NPY Y1 receptor antagonists [J]. European Journal of Pharmacology, 2002, 451(3): 245−256. doi: 10.1016/S0014-2999(02)02224-0
[16]	LIN Y T, HUANG Y L, TSAI S C, et al. Ablation of NPFFR2 in mice reduces response to single prolonged stress model [J]. Cells, 2020, 9(11): 2479. doi: 10.3390/cells9112479
[17]	ANKÖ M L, PANULA P. Regulation of endogenous human NPFF₂ receptor by neuropeptide FF in SK-N-MC neuroblastoma cell line [J]. Journal of Neurochemistry, 2006, 96(2): 573−584. doi: 10.1111/j.1471-4159.2005.03581.x
[18]	BURGESS D J. NF-κB shows its beneficial side [J]. Nature Reviews Cancer, 2011, 11(12): 832−833. doi: 10.1038/nrc3168
[19]	WU X F, LIU Y, GAO C F, et al. Novel alternative splicing variants of ACOX1 and their differential expression patterns in goats [J]. Archives Animal Breeding, 2018, 61(1): 59−70. doi: 10.5194/aab-61-59-2018
[20]	王芬, 贾建平, 秦伟, 等. GSK3B基因3′-UTR多态性与阿尔茨海默病发病风险的相关性分析 [J]. 脑与神经疾病杂志, 2019, 27(3):138−142. WANG F, JIA J P, QIN W, et al. Association of the polymorphisms in the 3′-UTR of GSK3B with the risk for Alzheimer's disease [J]. Journal of Brain and Nervous Diseases, 2019, 27(3): 138−142.(in Chinese)
[21]	李素雅, 张建宏, 陈文, 等. 鸡Lpin1基因3′-UTR遗传变异及其对miRNA结合位点的潜在效应 [J]. 中国农业科学, 2012, 45(8):1613−1620. doi: 10.3864/j.issn.0578-1752.2012.08.017 LI S Y, ZHANG J H, CHEN W, et al. Genetic variation analysis of 3′-UTR region of chicken Lpin1 gene and the potential effect on miRNA binding sites [J]. Scientia Agricultura Sinica, 2012, 45(8): 1613−1620.(in Chinese) doi: 10.3864/j.issn.0578-1752.2012.08.017
[22]	陈安利, 夏定国, 裘智勇, 等. 家蚕卵黄膜蛋白基因BmVMP23的3′-UTR变异对其表达的影响 [J]. 蚕业科学, 2013, 39(1):28−34. CHEN A L, XIA D G, QIU Z Y, et al. Influence of 3′-UTR mutation on expression of silkworm vitelline membrane protein gene BmVMP23 [J]. Science of Sericulture, 2013, 39(1): 28−34.(in Chinese)
[23]	杨韩, 张阳海, 王敏, 等. 陕北白绒山羊POU1F1基因3′-UTR多态性及其与生长性状的相关分析 [J]. 农业生物技术学报, 2019, 27(7):1224−1232. YANG H, ZHANG Y H, WANG M, et al. Polymorphisms of 3′-UTR of POU1F1 gene and its association with growth traits in Shaanbei white cashmere goats(Capra hircus) [J]. Journal of Agricultural Biotechnology, 2019, 27(7): 1224−1232.(in Chinese)
[24]	孙渭博, 张利平, 郎侠, 等. 四个绵羊品种BMPR-IB基因3′-UTR区多态性及其与胎产羔数的相关分析 [J]. 农业生物技术学报, 2020, 28(4):702−710. SUN W B, ZHANG L P, LANG X, et al. Polymorphism of 3′-UTR region of BMPR-IB gene and its correlation with litter size in four sheep(Ovis aries) varieties [J]. Journal of Agricultural Biotechnology, 2020, 28(4): 702−710.(in Chinese)