兰州鲇<i>NPY</i>基因的克隆和表达分析

doi:10.7668/hbnxb.20193911

华北农学报 ›› 2023, Vol. 38 ›› Issue (5): 227-238. doi: 10.7668/hbnxb.20193911

• 畜牧·水产·兽医 • 上一篇

兰州鲇NPY基因的克隆和表达分析

赖章龙¹, 阮超岭¹^,²^,⁶, 肖伟²^,³^,⁴^,⁵, 刘彦斌²^,³^,⁴^,⁵, 俞兆曦²^,³^,⁴, 刘凯²^,³^,⁴, 赛清云²^,³^,⁴, 田永华²^,³^,⁴, 张锋²^,³^,⁴^,⁵, 杨瑞兰³, 柳婷³, 杨立强³, 王玉涛¹^,⁶, 连总强¹^,²^,³^,⁴

¹ 喀什大学生命与地理科学学院,新疆喀什 844000
² 宁夏回族自治区水产研究所(有限公司),宁夏银川 750001
³ 甘肃农业大学动物科学技术学院,甘肃兰州 730070
⁴ 宁夏渔业工程技术研究中心,宁夏银川 750001
⁵ 宁夏渔业科技院士工作站,宁夏银川 750001
⁶ 新疆帕米尔高原生物资源与生态重点实验室,新疆喀什 844000

收稿日期:2022-12-08 出版日期:2023-10-28
通讯作者:
连总强(1980-),男,甘肃靖远人,高级工程师,博士,主要从事鱼类遗传育种与繁殖研究。王玉涛(1979-),男,甘肃镇原人,教授,博士,主要从事动物生态与分子进化研究。
连总强(1980-),男,甘肃靖远人,高级工程师,博士,主要从事鱼类遗传育种与繁殖研究。王玉涛(1979-),男,甘肃镇原人,教授,博士,主要从事动物生态与分子进化研究。
作者简介:
赖章龙(1994-),男,江西赣州人,硕士,主要从事鱼类遗传育种与繁殖研究。
基金资助:
国家重点研发计划项目(2018YFD0901202); 宁夏重点研发计划项目(2017BN06); 农业农村部财政专项(HHDC-2022-02); 2022年自治区重点研发计划黄河流域生态环境保护与高质量发展科技支撑专项

Molecular Cloning and Expression Analysis of NPY Gene in Silurus lanzhouensis

LAI Zhanglong¹, RUAN Chaoling¹^,²^,⁶, XIAO Wei²^,³^,⁴^,⁵, LIU Yanbin²^,³^,⁴^,⁵, YU Zhaoxi²^,³^,⁴, LIU Kai²^,³^,⁴, SAI Qingyun²^,³^,⁴, TIAN Yonghua²^,³^,⁴, ZHANG Feng²^,³^,⁴^,⁵, YANG Ruilan³, LIU Ting³, YANG Liqiang³, WANG Yutao¹^,⁶, LIAN Zongqiang¹^,²^,³^,⁴

¹ The College of Life and Geographic Sciences,Kashi University,Kashi 844000,China
² Ningxia Fisheries Research Institute,Yinchuan 750001,China
³ College of Animal Science and Technology,Gansu Agricultural University,Lanzhou 730070,China
⁴ Ningxia Fishery Engineering Technology Research Center,Yinchuan 750001,China
⁵ Academician Workstation of Ningxia Fishery Science and Technology, Yinchuan 750001,China
⁶ Key Laboratory of Biological Resources and Ecology of Pamirs Plateau in Xinjiang Uygur Autonomous Region,Kashi 844000,China

Received:2022-12-08 Published:2023-10-28

摘要/Abstract

摘要：

针对兰州鲇人工驯食转化率、苗种培育成活率低等问题,为利用基因编辑技术创制和培育易驯食兰州鲇新种质,开展了兰州鲇神经肽Y基因(NPY)的克隆及其表达分析研究。结果表明:兰州鲇NPY基因cDNA序列全长为775 bp,包括288 bp的开放阅读框,编码95个氨基酸,含有1个跨膜螺旋和1个信号肽胰腺激素类识别结合位点。兰州鲇NPY蛋白序列与南方鲇的一致性最高,为98.95%。荧光定量PCR检测发现NPY mRNA在脑组织中表达量最高,其次是下丘脑,肌肉中表达量最低。不同年龄雄性各组织中NPY mRNA表达水平均高于雌性;且雌雄性别组间均表现出随年龄的增长NPY mRNA表达下降的特征。利用原位杂交技术进行脑组织细胞定位发现,脑细胞中NPY主要在细胞质中表达;不同部位脑组织中主要以前脑的视前区、视顶盖和脑垂体,小脑的豆状核区域以及延脑的尾状核区域表达量较丰富。进一步进行饥饿补偿验证发现,饥饿可引起脑组织NPY表达量显著升高,摄食后NPY表达量显著下降。上述结果表明,NPY基因可能在兰州鲇生长和摄食过程中都具有重要作用,可作为对后续功能基因进行编辑的重要验证并创制新种质,对加快兰州鲇良种新品种选育具有重要的理论意义和技术支撑作用。

关键词: 兰州鲇, NPY基因, 生物信息学, 基因克隆, 原位杂交, 组织表达

Abstract:

In view of the low conversion rate of artificial domestication and survival rate of seedling cultivation of Silurus lanzhouensis,in order to use gene editing technology to create and cultivate new germplasm of S.lanzhouensis that was easy to domesticate,the effects of neuropeptide Y gene(NPY) of S.lanzhouensis were studied.The results showed that the full cDNA sequence of the NPY gene was 775 bp in length,with an open reading frame(ORF)of 288 bp,which encoded 95 amino acids and contained a transmembrane helix and a signal peptide binding site for pancreatic hormone recognition.The NPY protein sequence of S.lanzhouensis had the highest consistency with that of Silurus meridionalis,which was 98.95%.The Real-time Fluorescent Quantitative PCR results showed that NPY mRNA expression was the highest in brain tissue,followed by the hypothalamus,and the lowest in muscle.The expression level of NPY mRNA in male tissues of different ages was higher than that in female tissues;and the expression of NPY mRNA decreased with age in both sexes.Localization of brain cells by in situ hybridization found that NPY was mainly expressed in the cytoplasm,and abundant expression was found in the preoptic area,tectum,pituitary,the lentiform nucleus of cerebellum and the caudate nucleus of the medulla oblongata in different parts of the brain.Further verification of starvation compensation showed that the expression of NPY in brain tissue increased significantly after starvation,and decreased significantly after feeding.The above results showed that NPY gene might play an important role in the growth and feeding process of S.lanzhouensis.It could be used as an important verification of the subsequent functional gene editing and the creation of new germplasm,and had important theoretical significance and technical support for accelerating the breeding of new varieties of S.lanzhouensis.

Key words: Silurus lanzhouensis, NPY gene, Bioinformatics, Gene cloning, In situ hybridization, Tissue expression

赖章龙, 阮超岭, 肖伟, 刘彦斌, 俞兆曦, 刘凯, 赛清云, 田永华, 张锋, 杨瑞兰, 柳婷, 杨立强, 王玉涛, 连总强. 兰州鲇NPY基因的克隆和表达分析[J]. 华北农学报, 2023, 38(5): 227-238. doi: 10.7668/hbnxb.20193911.

LAI Zhanglong, RUAN Chaoling, XIAO Wei, LIU Yanbin, YU Zhaoxi, LIU Kai, SAI Qingyun, TIAN Yonghua, ZHANG Feng, YANG Ruilan, LIU Ting, YANG Liqiang, WANG Yutao, LIAN Zongqiang. Molecular Cloning and Expression Analysis of NPY Gene in Silurus lanzhouensis[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(5): 227-238. doi: 10.7668/hbnxb.20193911.

http://www.hbnxb.net/CN/Y2023/V38/I5/227

图/表 10

表1 本研究所用引物

Tab.1 Primers used in this study

引物名称 Primer name	序列(5'-3') Sequence (5'-3')	用途 Usage
NPY-5gsp1	TGTATCCTGATGCTTGTTACAGATG	5'RACE-PCR
NPY-5gsp2	GTAACCATTTCTGATAATGTCATTATGAT	5'RACE-PCR
NPY-3gsp1	TTTGTGATCTCATATGTAGTCCATCTGT	5'RACE-PCR
NPY-3gsp2	CCATCTGTAACAAGCATCAGGATAC	5'RACE-PCR
UPM	CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT	RACE通用引物
UPM short primer	CTAATACGACTCACTATAGGGC	RACE通用引物
NPY-qF	TCCTGCTGGTTGTATGTTTGGGTG	荧光定量PCR
NPY-qR	TGCCTCGTGATGAGGTTGATGTAG	荧光定量PCR
β-actin F	AAGATCATTGCCCCACCTGA	荧光定量PCR
β-actin R	CCTGCTTGCTGATCCACATC	荧光定量PCR
NPY-F	AACTAACTATGCGTCCTCG	中间序列克隆
NPY-R	TTGGTGTAGCCTTGTGCC	中间序列克隆
	AGACGCACACGTTTGCAAGAAAAC
	AACCAGCAGGATGCACGCGAC
	GGATATCCTTCCACCACTGCACC
	CCCCTCTCCTGGGTTCTGCGG
NPY-Mix	CGCAGTGTAATACTTCGCTAGATCCTCA	原位杂交
	CCTCGTGATGAGGTTGATGTAGTGTCT
	ATCAGCGTTTGACCTTTTACCATACC
	CCAAACAGCAGGTCTGGGGATAA
	ATATCTGGACCGCAGTTGCATCTC

图1 兰州鲇NPY基因扩增产物电泳

Fig.1 Electrophoresis result of amplified product of NPY in S.lanzhouensis M.DNA Marker DL3000。

图2 兰州鲇和人NPY基因共线性 Has.人;Sla.兰州鲇。

Fig.2 Syntenic analysis for NPY genes in S.lanzhouensis and H.sapiens Has.Homo sapiens; Sla.Silurus lanzhouensis.

图3 兰州鲇和其他硬骨鱼类NPY基因共线性 Sla.兰州鲇;Ipu.斑点叉尾鮰;Dre.斑马鱼;Cau.金鱼。

Fig.3 Syntenic analysis for NPY genes in S.lanzhouensis and other teleost species Sla.Silurus lanzhouensis;Ipu.Ictalurus punctatus;Dre.Danio rerio;Cau.Carassius auratus.

图4 兰州鲇NPY基因氨基酸序列同源性比对

Fig.4 Amino acid sequence homology of NPY gene of S.lanzhouensis

图5 NPY基因系统进化树

Fig.5 NPY gene phylogenetic tree

图6 NPY基因组织分布表达不同字母表示差异显著(P<0.05);相同字母表示差异不显著(P>0.05)。

Fig.6 Tissue distribution and expression of NPY gene Different letters mean significant differences at P<0.05;the same letters mean no significant differences at P>0.05.

图7 NPY基因在兰州鲇不同年龄雌雄间的表达水平 1.1龄;2.2龄;3.3龄;不同字母表示不同年龄段所有性别之间差异显著(P<0.05),相同字母表示差异不显著(P>0.05)。

Fig.7 Expression of NPY gene in the male and female S.lanzhouensis at different ages 1.1-year-old;2.2-year-old;3.3-year-old;different letters indicated that there were significant differences among all sexes in different age groups(P<0.05),while the same letters indicated that there was no significant difference(P>0.05).

图8 NPY基因在兰州鲇脑的细胞定位 A.NPY mRNA在脑冠状切面的细胞定位;B.NPY mRNA在脑失状切面的细胞定位;C.A图箭头处局部放大图;D.B图箭头处局部放大图。

Fig.8 The cellular localization of NPY gene in the brain of S.lanzhouensis A.Cellular localization of NPY mRNA in the coronal section of the brain;B.Cellular localization of NPY mRNA in the disappearing section of the brain;C.Partial magnification at the arrow of Fig.A;D.Partial magnification at the arrow of Fig.B.

图9 兰州鲇NPY mRNA在饥饿再摄食条件下的表达 CK.对照组;R.再摄食;不同字母表示不同处理时间差异显著(P<0.05),相同字母表示差异不显著(P>0.05)。

Fig.9 Expression of NPY mRNA in S.lanzhouensis under the condition of starvation and refeeding CK.Control group;R.Re-feeding;different letters showed significant difference in different treatment time(P<0.05),while the same letter indicated no significant difference(P>0.05).

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