西藏桑桑牦牛<i>PFN3</i>基因克隆、生物信息学及组织表达分析

doi:10.7668/hbnxb.20195594

华北农学报 ›› 2026, Vol. 41 ›› Issue (2): 214-221. doi: 10.7668/hbnxb.20195594

所属专题： 畜牧；

• 畜牧·水产·兽医 • 上一篇下一篇

西藏桑桑牦牛PFN3基因克隆、生物信息学及组织表达分析

何文斌¹^,², 杨焱¹^,³, 洛桑顿珠⁴, 喇永福¹^,², 任稳稳¹^,², 马晓明¹^,², 张强⁴, 卓玛次仁⁵, 尼玛加措⁵, 平措占堆⁴, 梁春年¹^,²

¹ 中国农业科学院兰州畜牧与兽药研究所, 甘肃省牦牛繁育工程重点实验室, 甘肃兰州 730050
² 农业农村部青藏高原畜禽遗传育种重点实验室, 甘肃兰州 730050
³ 甘肃省肃南裕固族自治县农业农村局, 甘肃张掖 734400
⁴ 西藏自治区农牧科学院畜牧兽医研究所, 西藏拉萨 850004
⁵ 日喀则市昂仁县农业农村局, 西藏日喀则 857001

收稿日期:2025-03-20 出版日期:2026-05-06
通讯作者:
平措占堆(1974—),男,西藏拉萨人,副研究员,博士,主要从事牦牛遗传育种研究。
梁春年(1973—),男,甘肃武威人,研究员,博士,主要从事动物遗传育种与繁殖研究。
作者简介:
何文斌(1998—),男,甘肃秦州人,硕士,主要从事畜牧研究。
基金资助:
西藏自治区区域科技协同创新专项(QYATZX-RKZ2022-03); 西藏自治区重点研发计划(XZ202402ZY0012); 现代肉牛牦牛产业技术体系(CARS-37)

Cloning,Bioinformatics and Tissue Expression Analysis of PFN3 Gene in Sangsang Yak from Tibet

HE Wenbin¹^,², YANG Yan¹^,³, Lobsong Dunzhu⁴, LA Yongfu¹^,², REN Wenwen¹^,², MA Xiaoming¹^,², ZHANG Qiang⁴, Zhuoma Tsering⁵, Nima Gyatso⁵, Phuntsok Zhandui⁴, LIANG Chunnian¹^,²

¹ Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, Key Laboratory of Yak Breeding of Gansu Province, Lanzhou 730050, China
² Key Laboratory of Animal Genetics and Breeding on Tibetan Plateau, Ministry of Agriculture and Rural Affairs, Lanzhou 730050, China
³ Bureau of Agriculture and Rural Affairs of Sunan Yugur Autonomous County, Gansu Province, Zhangye 734400, China
⁴ Institute of Animal Science and Veterinary, Tibet Academy of Agricultural and Animal Husbandry Sciences, Lhasa 850004, China
⁵ Angren County Agricultural and Rural Bureau of Xigaze City, Xigaze 857001, China

Received:2025-03-20 Published:2026-05-06

摘要/Abstract

摘要：

旨在探究桑桑牦牛PFN3基因(Profilin 3)的结构与理化指标,并利用qPCR检测RPL8基因在各组织中的表达情况,为挖掘牦牛PFN3基因的生物学功能提奠定理论基础。以心脏组织cDNA为模板,克隆牦牛PFN3基因的CDS区序列并检测该基因在不同组织中的表达。并使用多种软件和在线工具进行生物信息学分析。结果表明:桑桑牦牛PFN3基因编码区全长为414 bp,共有3个开放阅读框,编码137个氨基酸;通过分析牦牛PFN3蛋白显示,脂肪族系数为101.75,理论等电点9.04,不稳定指数37.22,该蛋白属于稳定的疏水蛋白;该蛋白存在4处潜在磷酸化位点,没有信号肽和跨膜区;氨基酸组成中甘氨酸(Gly)占比最高,为14.6%;亚细胞定位发现,PFN3蛋白在细胞质、细胞核和线粒体内的占比分别为43.5%,30.4%和26.1%;同源性比对发现,桑桑牦牛与野牦牛同源性最近,与非洲疣猪和欧亚野猪的亲缘关系最远;对互作蛋白进行KEGG通路富集分析,发现这些蛋白主要富集在肌动蛋白细胞骨架调控、Rap1信号通路、聚焦黏附、沙门菌感染和肌萎缩侧索硬化等通路中。qPCR结果显示,桑桑牦牛PFN3基因在直肠组织中表达最高,显著高于其他组织,在心脏中表达最低。

关键词: 桑桑牦牛, PFN3基因, 克隆, 生物信息学分析, 组织表达

Abstract:

The aim of this study was to investigate the structure and physicochemical index of PFN3 gene(Profilin 3)in Sangsang yak,and to detect the expression of RPL8 gene in various tissues by qPCR,so as to lay a theoretical foundation for exploring the biological function of PFN3 gene in Sangsang yak.Using cardiac tissue cDNA as template,we cloned the CDS region of PFN3 gene in yak and detected its expression in different tissues.Bioinformatics analysis is performed using a variety of software and online tools.The results showed that the PFN3 coding region of Sangsang yak was 414 bp in length,with 3 open reading frames encoding 137 amino acids.The analysis of PFN3 protein in yak showed that the aliphatic coefficient was 101.75,the theoretical isoelectric point was 9.04,and the instability index was 37.22,indicating that PFN3 protein was a stable hydrophobic protein.The protein had 4 potential phosphorylation sites and no signal peptide and transmembrane region.Glycine(Gly)accounted for 14.6% of the total amino acids.Subcellular localization showed that PFN3 protein accounted for 43.5%,30.4% and 26.1% in cytoplasm,nucleus and mitochondria,respectively.Homology comparison showed that Sangsang yak had the closest homology with wild yak,and was the most closely related to African warthog and Eurasian boar.KEGG pathway enrichment analysis of interacting proteins showed that these proteins were mainly enriched in actin cytoskeleton regulation,Rap1 signaling pathway,focused adhesion,salmonella infection and amyotrophic lateral sclerosis.qPCR results showed that PFN3 gene expression in Sangsang yak was the highest in rectal tissue,significantly higher than that in other tissues,and the lowest in heart.

Key words: Sang mulberry yak, PFN3 gene, Clone, Bioinformatics and the analysis, Tissue expression

中图分类号:

S823.85

何文斌, 杨焱, 洛桑顿珠, 喇永福, 任稳稳, 马晓明, 张强, 卓玛次仁, 尼玛加措, 平措占堆, 梁春年. 西藏桑桑牦牛PFN3基因克隆、生物信息学及组织表达分析[J]. 华北农学报, 2026, 41(2): 214-221. doi: 10.7668/hbnxb.20195594.

HE Wenbin, YANG Yan, Lobsong Dunzhu, LA Yongfu, REN Wenwen, MA Xiaoming, ZHANG Qiang, Zhuoma Tsering, Nima Gyatso, Phuntsok Zhandui, LIANG Chunnian. Cloning,Bioinformatics and Tissue Expression Analysis of PFN3 Gene in Sangsang Yak from Tibet[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(2): 214-221. doi: 10.7668/hbnxb.20195594.

http://www.hbnxb.net/CN/Y2026/V41/I2/214

图/表 9

表1 PFN3引物序列信息

Tab.1 PFN3 gene primer sequence

基因 Gene	用途 Purpose	序列(5'—3') Sequence (5'—3')	退火温度/℃ Annealing temperature	目的产物长度/bp Length of the target product
PFN3	PCR	F1 ACTTCATAACCACGCCCCTG	57	567
		R1 CACGCCCAGGTCAGACTTTA
PFN3	qPCR	F2 TGGGCGACTGGAAAGGTTAC	58	82
		R2 TTGTCCGAGTGGCCCACAAT
GAPDH	qPCR	F2 TTGTGATGGGCGTGAACC	58	169
		R2 GTCTTCTGGGTGGCAGTGAT

表2 PFN3基因生物信息学方法

Tab.2 PFN3 gene bioinformatics method

软件 Software	用途 Application
NCBI BLAST(https://blast.ncbi.nlm.nih.gov/Blast.cgi)	序列对比验证
MEGA 10.1	构建系统进化树
Open reading frame finder(OFR Finder, http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi)	查看开放阅读框
ProtParam(http://www.expasy.ch/tools/Protparam.html)	蛋白质理化性质分析
ProtScale(https://web.expasy.org/protscale/)	蛋白亲疏水性分析
SignalP 4.1(http://www.cbs.dtu.dk/services/SignalP/)	信号肽预测
TMHMM(http://www.cbs.dtu.dk/services/TMHMM/)	跨膜结构域分析
Psort Ⅱ(https://wolfpsort.hgc.jp/)	蛋白亚细胞定位
NetPhos 3.1 Server(http://www.cbs.dtu.dk/services/NetPhos/)	磷酸化位点预测
Sopma(https://npsa-prabi.ibcp.fr/cgi- bin/npsa_automat.pl?page=npsa_sopma.html)	蛋白质二级结构预测
SWISS-MODEL(http://www.expasy.org/swissmod/SWISS-MODEL.html)	蛋白质三级结构预测
STRING(https://string-db.org/)	蛋白互作网络预测

图1 PFN3基因扩增电泳

Fig.1 Electrophoresis pattern of PFN3 gene amplification

图2 PFN3在各物种间的系统进化树分析 A.PFN3基因在各物种间构建的核苷酸系统进化树;B.PFN3在各物种间构建的氨基酸系统进化树。

Fig.2 Phylogenetic tree analysis of PFN3 among species A.Phylogenetic tree of PFN3 gene among different species;B.Amino acid phylogenetic tree constructed by PFN3 among species.

表3 PFN3蛋白中氨基酸含量

Tab.3 Amino acid composition of the PFN3 protein

氨基酸 Amino acid	数量/个 Quantity	含量/% Content	氨基酸 Amino acid	数量/个 Quantity	含量/% Content
丙氨酸Ala (A)	11	8.0	亮氨酸Leu (L)	16	11.7
精氨酸Arg (R)	14	10.2	赖氨酸Lys (K)	3	2.2
天冬酰胺Asn (N)	2	1.5	蛋氨酸Met (M)	3	2.2
天冬氨酸Asp (D)	10	7.3	苯丙氨酸Phe (F)	1	0.7
半胱氨酸Cys (C)	5	2.9	脯氨酸Pro (P)	4	2.9
谷氨酰胺Gln (Q)	4	2.9	丝氨酸Ser (S)	6	4.4
谷氨酸Glu (E)	3	2.2	苏氨酸Thr (T)	6	4.4
甘氨酸Gly (G)	20	14.6	色氨酸Trp (W)	2	1.5
组氨酸His (H)	6	4.4	酪氨酸Tyr (Y)	1	0.7
异亮氨酸Ile (I)	8	5.8	缬氨酸Val (V)	12	8.8

图3 PFN3蛋白跨膜结构域及磷酸化位点预测 A.PFN3蛋白的跨膜结构域预测;B.PFN3蛋白的磷酸化位点预测。

Fig.3 PFN3 protein transmembrane domains and phosphorylation site predictions A.Transmembrane domain prediction of PFN3 protein;B.Phosphorylation site prediction of PFN3 protein.

图4 PFN3蛋白高级结构预测 A.PFN3蛋白二级结构预测:h.α-螺旋,c.无规则卷曲,t.β-转角,e.延伸链;B.PFN3蛋白三级结构预测。

Fig.4 Prediction of PFN3 protein high-level structure A.PFN3 protein secondary structure prediction:h.α-helix,c.Random coil,t.β-turn,e.Extended strand;B.PFN3 protein tertiary structure prediction.

图5 PFN3蛋白质互作网络预测及KEGG通路富集分析 A.PFN3蛋白质互作网络预测;B.PFN3互作蛋白KEGG通路富集。

Fig.5 PFN3 protein interaction network prediction and KEGG pathway enrichment analysis A.PFN3 protein interaction network prediction;B.KEGG pathway enrichment of PFN3 interacting proteins.

图6 PFN3基因在桑桑牦牛不同组织中的表达水平分析不同小写字母表示各组织间差异显著(P<0.05)。

Fig.6 Expression level analysis of PFN3 gene in different tissues of Sangsang yak Different lowercase letters indicated significant differences among tissues(P<0.05).

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	Zhang Y Y, Zhao Y, Wang R, Qin C Y, Wang P, Wang S Y, Li F Q, Liu L X, Huo H L, Li W Z, Lu J, Huo J L. In Silico cloning,RT-PCR verification and tissues expression of novel swine gene PFN3[J]. Journal of Yunnan Agricultural University:Natural Science Edition, 2014, 29(2):179-186.

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西藏桑桑牦牛PFN3基因克隆、生物信息学及组织表达分析

Cloning,Bioinformatics and Tissue Expression Analysis of PFN3 Gene in Sangsang Yak from Tibet

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西藏桑桑牦牛PFN3基因克隆、生物信息学及组织表达分析

Cloning,Bioinformatics and Tissue Expression Analysis of PFN3 Gene in Sangsang Yak from Tibet

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