帕米尔牦牛<i>PPP1R11</i>基因克隆、生物信息学及组织表达分析

doi:10.7668/hbnxb.20195471

摘要/Abstract

摘要： 为研究牦牛PPP1R11基因的结构和功能,及在8个不同组织中的表达规律,以帕米尔牦牛睾丸组织cDNA为模板,使用PCR技术克隆帕米尔牦牛PPP1R11基因CDS区序列,并进行生物信息学分析,PPP1R11基因在帕米尔牦牛8个组织中的表达水平通过实时荧光定量PCR (RT-qPCR)进行测定。结果表明,帕米尔牦牛PPP1R11基因CDS区全长324 bp,编码107个氨基酸。同源性对比结果显示,帕米尔牦牛与野牦牛的相似性最高(100%),与小家鼠的相似性最低(84.6%)。生物信息学分析结果表明,PPP1R11 蛋白分子式为C₅₀₃H₈₀₃N₁₆₃O₁₆₄S₈,原子总数为1 641,不稳定指数和总平均亲水指数分别为65.91和-1.385,为亲水性蛋白;PPP1R11蛋白不具有信号肽和跨膜结构,主要定位在细胞核中;且该蛋白有1个糖基化位点和19处潜在的磷酸化位点;蛋白的二级结构以无规则卷曲为主,与PPP1R7、PPP1CA和NSFL1C等蛋白存在主要相互作用。RT-qPCR分析表明,PPP1R11基因在帕米尔牦牛心脏、肌肉、脂肪、脾脏、肝脏、胰脏、肾脏和睾丸这8个组织中存在差异性表达,相较于其他7种组织,PPP1R11在睾丸中的表达显著上调。

关键词: 帕米尔牦牛, PPP1R11基因, 基因克隆, 组织表达

Abstract:

To investigate the structure and function of the PPP1R11 gene in yaks,along with its expression pattern in eight distinct tissues,cDNA from the testicular tissue of Pamir yaks was utilized as the template,the CDS region sequence of the PPP1R11 gene in Pamir yaks was cloned via PCR technology,followed by bioinformatics analysis.The expression level of the PPP1R11 gene in eight tissues of the Pamir yak was determined using Real-time Fluorescence Quantitative PCR(RT-qPCR).The results showed that the coding sequence(CDS region)of PPP1R11 gene in Pamir yak was 324 bp and encoded 107 amino acids.The homology comparison showed that Pamir yak had the highest similarity with Bos mutus(100%)and the lowest similarity with Mus musculus(84.6%).The results of bioinformatics analysis showed that the molecular formula of PPP1R11 protein was C₅₀₃H₈₀₃N₁₆₃O₁₆₄S₈,the total number of atoms was 1 641,the instability index and the total average hydrophilic index were 65.91 and -1.385,respectively.PPP1R11 protein had no signal peptide and transmembrane structure,and was mainly located in the nucleus.The protein had one glycosylation site and 19 potential phosphorylation sites.The secondary structure of the protein was mainly random curling,and it mainly interacted with PPP1R7,PPP1CA and NSFL1C.The RT-qPCR results showed that PPP1R11 gene was expressed differently in heart,muscle,fat,spleen,liver,pancreas,kidney and testis of Pamir yak,and the expression level of PPP1R11 gene was significantly higher in testis than in the other seven tissues.

Key words: Pamir yak, PPP1R11 gene, Gene cloning, Tissue expression

中图分类号:

邓婧瑛, 于沁冉, 王佟, 李宁, 贾建磊, 包鹏甲, 阎萍. 帕米尔牦牛PPP1R11基因克隆、生物信息学及组织表达分析[J]. 华北农学报, 2025, 40(6): 194-201. doi: 10.7668/hbnxb.20195471.

DENG Jingying, YU Qinran, WANG Tong, LI Ning, JIA Jianlei, BAO Pengjia, YAN Ping. Cloning,Bioinformatics and Tissue Expression Analysis of PPP1R11 Gene in Pamir Yak[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(6): 194-201. doi: 10.7668/hbnxb.20195471.

http://www.hbnxb.net/CN/Y2025/V40/I6/194

图/表 14

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基因 Gene	引物序列(5'—3') Primer sequences(5'—3')	片段大小/bp Fragment size	退火温度/℃ Annealing temperature	用途 Application
PPP1R11	F:GGGTTTGGGAGAATCGCAGA	752	57.0	PCR
	R:ATCCCAGGACAGGCAGTTTG
PPP1R11	F:AAAGGTGGAATGGACGAGTGA	70	60.0	RT-qPCR
	R:ACAGCAGCATTTTGATGAGCG
GAPDH	F:GGCATCGTGGAGGGACTTATG	198	60.0	RT-qPCR
	R:CCAGTGAGCTTCCCGTTGAG

基因 Gene	引物序列(5'—3') Primer sequences(5'—3')	片段大小/bp Fragment size	退火温度/℃ Annealing temperature	用途 Application
PPP1R11	F:GGGTTTGGGAGAATCGCAGA	752	57.0	PCR
	R:ATCCCAGGACAGGCAGTTTG
PPP1R11	F:AAAGGTGGAATGGACGAGTGA	70	60.0	RT-qPCR
	R:ACAGCAGCATTTTGATGAGCG
GAPDH	F:GGCATCGTGGAGGGACTTATG	198	60.0	RT-qPCR
	R:CCAGTGAGCTTCCCGTTGAG

软件名称 Software name	网址 Website	功能 Function
ORF Finder	https://www.ncbi.nlm.nih.gov/orffinder/	开放阅读框分析
NetNGlyc-1.0	https://services.healthtech.dtu.dk/services/NetNGlyc-1.0/	蛋白质糖基化位点预测
TMHMM	https://services.healthtech.dtu.dk/service/TMHMM-2.0	蛋白质跨膜结构预测
Psort	https://wolfpsort.hgc.jp/	蛋白质亚细胞定位预测
Sopma	https://npsa.lyon.inserm.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html	蛋白质二级结构预测
SWISS-MODEL	https://swissmodel.expasy.org	蛋白质三级结构预测
ProtParam	https://www.expasy.org/tools/Protparam.html	蛋白质理化性质分析
NetPhos 2.0	https://services.healthtech.dtu.dk/services/NetPhos-3.1/	氨基酸磷酸化位点预测
STRING	https://cn.stving-db.org	蛋白质互作网络预测
ProtScale	https://web.expasy.org/protscale/	蛋白质亲疏水性分析
SignalP 4.1	https://services.healthtech.dtu.dk/services/SignalP-4.1/	蛋白质信号肽分析
Blast	https://blast.ncbi.nlm.nih.gov/Blast.cgi	同源性比对分析

软件名称 Software name	网址 Website	功能 Function
ORF Finder	https://www.ncbi.nlm.nih.gov/orffinder/	开放阅读框分析
NetNGlyc-1.0	https://services.healthtech.dtu.dk/services/NetNGlyc-1.0/	蛋白质糖基化位点预测
TMHMM	https://services.healthtech.dtu.dk/service/TMHMM-2.0	蛋白质跨膜结构预测
Psort	https://wolfpsort.hgc.jp/	蛋白质亚细胞定位预测
Sopma	https://npsa.lyon.inserm.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html	蛋白质二级结构预测
SWISS-MODEL	https://swissmodel.expasy.org	蛋白质三级结构预测
ProtParam	https://www.expasy.org/tools/Protparam.html	蛋白质理化性质分析
NetPhos 2.0	https://services.healthtech.dtu.dk/services/NetPhos-3.1/	氨基酸磷酸化位点预测
STRING	https://cn.stving-db.org	蛋白质互作网络预测
ProtScale	https://web.expasy.org/protscale/	蛋白质亲疏水性分析
SignalP 4.1	https://services.healthtech.dtu.dk/services/SignalP-4.1/	蛋白质信号肽分析
Blast	https://blast.ncbi.nlm.nih.gov/Blast.cgi	同源性比对分析

标注 Label	链 Strand	框架 Frame	起点/ bp Start	终点/ bp Stop	ORF 长度/bp ORF length	氨基酸长度/aa Amina acid length
ORF1	+	1	<1	324	324	107
ORF2	+	2	92	>322	231	76
ORF3	—	1	243	115	129	42
ORF4	—	2	101	>3	99	32
ORF5	—	3	115	32	84	27

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帕米尔牦牛PPP1R11基因克隆、生物信息学及组织表达分析

Cloning,Bioinformatics and Tissue Expression Analysis of PPP1R11 Gene in Pamir Yak

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氨基酸种类 Amino acid species	数量/个 Quantity	含量/% Content	氨基酸种类 Amino acid species	数量/个 Quantity	含量/% Content
丙氨酸Ala (A)	2	1.9	亮氨酸Leu (L)	2	1.9
精氨酸 Arg(R)	10	9.3	赖氨酸Lys (K)	8	7.5
天冬酰胺Asn (N)	2	1.9	蛋氨酸Met (M)	3	2.8
天冬氨酸Asp (D)	5	4.7	苯丙氨酸Phe (F)	1	0.9
半胱氨酸Cys (C)	5	4.7	脯氨酸Pro (P)	14	13.1
谷氨酰胺Gln (Q)	3	2.8	丝氨酸Ser (S)	11	10.3
谷氨酸Glu (E)	11	10.3	苏氨酸Thr (T)	7	6.5
甘氨酸Gly (G)	8	7.5	色氨酸Trp (W)	1	0.9
组氨酸His (H)	6	5.6	酪氨酸Tyr (Y)	1	0.9
异亮氨酸Ile (I)	2	1.9	缬氨酸Val (V)	5	4.7

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帕米尔牦牛PPP1R11基因克隆、生物信息学及组织表达分析

Cloning,Bioinformatics and Tissue Expression Analysis of PPP1R11 Gene in Pamir Yak

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