牦牛<i>PID1</i>基因克隆、生物信息学及组织表达分析

doi:10.7668/hbnxb.20194907

华北农学报 ›› 2024, Vol. 39 ›› Issue (6): 216-223. doi: 10.7668/hbnxb.20194907

所属专题： 畜牧；

• 畜牧·水产·兽医 • 上一篇下一篇

牦牛PID1基因克隆、生物信息学及组织表达分析

余道宁¹^,²^,³, 王佟¹^,²^,³, 洛桑顿珠⁴, 平措占堆⁴, 张强⁴, 卓玛次仁⁵, 尼玛加措⁵, 张德荣¹, 梁春年²^,³

¹ 西北民族大学生命科学与工程学院,甘肃兰州 730124
² 中国农业科学院兰州畜牧与兽药研究所,甘肃省牦牛繁育重点实验室,甘肃兰州 730050
³ 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
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:2024-05-27 出版日期:2024-12-28
通讯作者:
张德荣(1979—),男,甘肃天祝人,讲师,博士,主要从事动物遗传育种与繁殖研究。
梁春年(1973—),男,甘肃武威人,研究员,博士,博士生导师,主要从事动物遗传育种与繁殖研究。
作者简介:
余道宁(1999—),男,河南信阳人,在读硕士,主要从事动物遗传育种与繁殖研究。
基金资助:
西藏自治区区域科技协同创新项目(QYXTZX-RKZ2022-03); 国家重点研发计划项目(2022YFD1302101); 现代肉牛牦牛产业技术体系项目(CARS-37)

Cloning,Bioinformatics and Tissue Expression Analysis of PID1 Gene in Yak

YU Daoning¹^,²^,³, WANG Tong¹^,²^,³, LOBSANG Dondrub⁴, PINGCUO Zhandui⁴, ZHANG Qiang⁴, ZHUOMA Ciren⁵, NIMA Jiacuo⁵, ZHANG Derong¹, LIANG Chunnian²^,³

¹ Life Science and Engineering College,Northwest Minzu University,Lanzhou 730124,China
² Lanzhou Institute of Husbandry and Pharmaceutical Sciences of Chinese Academy of Agricultural Sciences,Key Laboratory of Yak Breeding,Lanzhou 730050,China
³ Key Laboratory of Animal Genetics and Breeding on Tibetan Plateau,Ministry of Agriculture and Rural Affairs,Lanzhou 730050,China
⁴ Institute of Animal Husbandry and Veterinary,Tibet Autonomous Regional Academy of Agricultural Sciences,Lhasa 850004,China
⁵ Agriculture and Rural Bureau of Ngamring County,Xigaze City,Xigaze 857001,China

Received:2024-05-27 Published:2024-12-28

摘要/Abstract

摘要：

为研究牦牛磷酸酪氨酸互作结构域1基因(PID1)的结构及功能,并探究其在各组织中的表达情况,以桑桑牦牛脂肪组织cDNA为模板克隆桑桑牦牛PID1基因CDS区序列,并对其进行生物信息学分析。同时,采用实时荧光定量PCR方法(RT-qPCR),对牦牛的7个不同组织包括心、肝、脾、肺、肾、背部肌肉及脂肪进行PID1基因表达水平的定量。结果表明,牦牛PID1基因的编码区长度是654 bp,编码217个氨基酸;通过同源性比对,结果发现,牦牛与野牦牛之间的亲缘关系最为接近,相似度达到100%;经过蛋白质分析发现,牦牛PID1蛋白的分子质量约为24.84 ku,理论等电点为6.30。根据不稳定系数计算结果显示,其不稳定性较高(47.96),属于一种不稳定蛋白质。该蛋白内含有1个N-糖基化位点和23个磷酸化位点,无信号肽和跨膜结构。RT-qPCR试验结果表明,在各个组织中都能检测到PID1基因的表达情况,其中在肺部表达量最高。该研究克隆了牦牛PID1基因,并对其蛋白结构进行了分析,同时还研究了该基因在牦牛组织中的表达情况。为进一步探究PID1基因在牦牛脂肪沉积过程中的作用提供了初步数据。

关键词: 牦牛, PID1基因, 基因克隆, 生物信息学

Abstract:

To study the structure and function of phosphotyrosine interaction domain 1 (PID1) gene in yak,and to explore its expression in various tissues.The CDS region of Sangsang yak PID1 gene was cloned using Sangsang yak adipose tissue cDNA as template,and the sequence was analyzed bioinformatically.Meanwhile,the relative expression level of PID1 gene was detected in seven tissues of yak namely heart,liver,spleen,lung,kidney,longissimus dorsi muscle and adipose tissue by Real-time Fluorescence Quantitative PCR(RT-qPCR).The results showed that the PID1 gene in yaks had a coding region length of 654 bp,which encoded 217 amino acids.Homology comparison showed that yak and wild yak were closely related,and the similarity reached 100%.The molecular weight of yak PID1 protein was about 24.84 ku and the theoretical isoelectric point was 6.30.According to the calculation results of instability coefficient,the instability of the protein was high (47.96),and it belonged to an unstable protein.The protein had one N-glycosylation site and 23 phosphorylation sites,with no signal peptide or transmembrane structure.The results of RT-qPCR showed that the expression of PID1 gene could be detected in all tissues,with the highest expression in the lung.The yak PID1 gene was cloned and its protein structure was analyzed.The expression of PID1 gene in yak tissue was also studied.Further study on the role of PID1 gene in yak fat deposition provided preliminary data.

Key words: Yak, PID1 gene, Gene cloning, Bioinformatics

余道宁, 王佟, 洛桑顿珠, 平措占堆, 张强, 卓玛次仁, 尼玛加措, 张德荣, 梁春年. 牦牛PID1基因克隆、生物信息学及组织表达分析[J]. 华北农学报, 2024, 39(6): 216-223. doi: 10.7668/hbnxb.20194907.

YU Daoning, WANG Tong, LOBSANG Dondrub, PINGCUO Zhandui, ZHANG Qiang, ZHUOMA Ciren, NIMA Jiacuo, ZHANG Derong, LIANG Chunnian. Cloning,Bioinformatics and Tissue Expression Analysis of PID1 Gene in Yak[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(6): 216-223. doi: 10.7668/hbnxb.20194907.

http://www.hbnxb.net/CN/Y2024/V39/I6/216

图/表 14

表1 PID1基因引物序列

Tab.1 Primer sequence of PID1 gene

基因 Gene	引物序列(5'—3') Primer sequences(5'—3')	产物长度/bp Product size	退火温度/℃ Annealing temperature	用途 Application
PID1	F:CCCATCGACAGAGTCTTGCC	750	60.0	PCR
	R:TTCCTTGACCAATGCTGCCT
PID1	F:TCTTTCCGGCTAATGCCCTC	153	59.0	RT-qPCR
	R:AGATGTTGGGGCTCACGTTG
GAPDH	F:GGCATCGTGGAGGGACTTATG	186	59.0	RT-qPCR
	R:GCCAGTGAGCTTCCCGTTGAG

表2 生物信息学分析软件

Tab.2 Bioinformatics analysis software

工具 Online tools	功能 Analysis content
ORF Finder	开放阅读框分析
Blast	相似性比对分析
ProtParam	蛋白质理化性质分析
ProtScale	蛋白质亲疏水性分析
SignalP 4.1	蛋白质信号肽分析
TMHMM 2.0	蛋白质跨膜结构预测
NetPhos 3.1	蛋白磷酸化位点预测
NetNGlyc	蛋白糖基化位点预测
SOPMA	蛋白质二级结构预测
SWISS-MODEL	蛋白质三级结构预测
STRING	蛋白互作网络预测

图1 牦牛PID1基因PCR扩增结果 M.GL DNA Marker 1000;1.PCR扩增产物。

Fig.1 PCR amplification results of PID1 gene M.GL DNA Marker 1000;1.PCR amplification product.

图2 PID1基因序列同源性分析

Fig.2 Sequence homology analysis of PID1 gene

图3 PID1基因系统进化树

Fig.3 PID1 gene phylogenetic tree

表3 牦牛PID1蛋白氨基酸组成

Tab.3 Amino acid composition of yak PID1 protein

氨基酸种类 Amino acid	占比/% Proportion	氨基酸种类 Amino acid	占比/% Proportion
丙氨酸Ala (A)	7.8	亮氨酸Leu (L)	9.2
精氨酸Arg (R)	3.7	赖氨酸Lys (K)	7.8
天冬酰胺Asn (N)	2.8	甲硫氨酸Met (M)	4.1
天冬氨酸Asp (D)	4.6	苯丙氨酸Phe (F)	4.1
半胱氨酸Cys (C)	2.8	脯氨酸Pro (P)	4.6
谷氨酰胺Gln (Q)	4.1	丝氨酸Ser (S)	6.0
谷氨酸Glu (E)	8.8	苏氨酸Thr (T)	7.8
甘氨酸Gly (G)	3.2	色氨酸Trp (W)	1.8
组氨酸His (H)	5.5	酪氨酸Tyr (Y)	1.8
异亮氨酸Ile (I)	3.7	缬氨酸Val (V)	5.5

图4 牦牛PID1蛋白疏水性分析

Fig.4 Hydrophobicity analysis of yak PID1 protein

图5 蛋白信号肽预测

Fig.5 Protein signal peptide prediction

图6 蛋白跨膜结构预测

Fig.6 Prediction of protein transmembrane structure

图7 牦牛PID1蛋白N-糖基化位点分析

Fig.7 Prediction of N-glycosylation sites in yak PID1 protein

图8 牦牛PID1蛋白磷酸位点分析

Fig.8 Analysis of phosphate sites in yak PID1 protein

图9 牦牛PID1蛋白二(A)、三级(B)结构预测

Fig.9 Yak PID1 protein secondary(A)and tertiary(B)structure prediction

图10 牦牛PID1蛋白互作网络分析

Fig.10 Analysis of protein-protein interaction network in yak PID1

图11 牦牛PID1基因在不同组织中的相对表达不同大写字母表示不同组织间差异极显著(P<0.01)。

Fig.11 Relative expression of yak PID1 gene in different tissues Different capital letters denote extremely significant differences between different organs (P<0.01).

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[26]	Xu H G, Xu G Y, Wang D H, Zheng C L, Wan L. Molecular cloning and tissue distribution of the phosphotyrosine interaction domain containing 1 (PID1) gene in Tianfu goat[J]. Gene, 2013, 515(1):71-77.doi:10.1016/j.gene.2012.11.025. pmid: 23237778
[27]	钱源, 曾勇庆, 杜金芳, 崔景香, 李华, 陈其美, 宋一萍, 陈伟. 猪PID1基因CDS区的克隆及其mRNA表达与肌内脂肪沉积关系[J]. 遗传, 2010, 32(11):1153-1158.doi:10.3724/SP.J.1005.2010.01153.
	Qian Y, Zeng Y Q, Du J F, Cui J X, Li H, Chen Q M, Song Y P, Chen W. CDS cloning and relationship between intramuscular fat content and mRNA expression of PID1 gene in pig[J]. Hereditas, 2010, 32(11):1153-1158.
[28]	栾兆进, 贺建宁, 程明, 刘开东, 曲绪仙, 柳楠. 绵羊肌肉组织MyoG和PID1基因的表达及其与肌内脂肪含量的关系[J]. 畜牧兽医学报, 2016, 47(10):1986-1994.doi:10.11843/j.issn.0366-6964.2016.10.006.
	Luan Z J, He J N, Cheng M, Liu K D, Qu X X, Liu N. Association of the MyoG and PID1 gene expression with intramuscular fat content in sheep muscles[J]. Acta Veterinaria et Zootechnica Sinica, 2016, 47(10):1986-1994.
[29]	徐正刚, 杨伦, 曾勇庆, 张哲, 陈伟, 杨云, 房国锋, 王守栋. RNA干扰沉默PID1基因肉兔模型的构建[J]. 畜牧兽医学报, 2014, 45(5):750-756.doi:10.11843/j.issn.0366-6964.2014.05.010.
	Xu Z G, Yang L, Zeng Y Q, Zhang Z, Chen W, Yang Y, Fang G F, Wang S D. Construction of transgenic rabbit model by RNA interferencing PID1 gene[J]. Acta Veterinaria et Zootechnica Sinica, 2014, 45(5):750-756.

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