美仁牦牛PHB基因克隆及组织表达

doi:10.7668/hbnxb.20194168

华北农学报 ›› 2023, Vol. 38 ›› Issue (5): 218-226. doi: 10.7668/hbnxb.20194168

所属专题： 畜牧；

• 畜牧·水产·兽医 • 上一篇下一篇

美仁牦牛PHB基因克隆及组织表达

杨国武¹^,²^,³, 王佟¹^,²^,³, 马小勇²^,³, 扎老⁴, 路建卫⁴, 赵雪⁵, 阎萍²^,³, 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,甘肃省牦牛繁育工程重点实验室, 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 合作市佐盖多玛乡畜牧站, 甘肃合作 747000
⁵ 合作市农畜产品质量安全检测检验中心,甘肃合作 747000

收稿日期:2023-04-06 出版日期:2023-10-28
通讯作者:
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:
杨国武(1997-),男,宁夏固原人,在读硕士,主要从事动物遗传育种与繁殖研究。
基金资助:
国家重点研发计划(2021YFD1600200); 甘肃省基础研究创新群体项目(20JR5RA580); 合作市牦牛种质提升与提质增效项目; 牦牛现代肉牛牦牛产业技术体系(CARS-37); 中国农业科学院创新工程(25-LIHPS-01); 甘肃省科技重大专项(21ZD10NA001); 甘肃省科技重大专项(GZGG-2021-1)

Cloning and Tissue Expression PHB Gene in Meiren Yak

YANG Guowu¹^,²^,³, WANG Tong¹^,²^,³, MA Xiaoyong²^,³, ZHA Lao⁴, LU Jianwei⁴, ZHAO Xue⁵, YAN Ping²^,³, 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 Engineering of Gansu Province,Lanzhou 730050,China
³ Key Laboratory of Animal Genetics and Breeding on the Tibetan Plateau,Ministry of Agriculture and Rural Affairs,Lanzhou 730050,China
⁴ Animal Husbandry Station of Zogaidoma Township,Hezuo 747000,China
⁵ Quality and Safety Inspection Center of Agricultural and Livestock Products in Hezuo,Hezuo 747000,China

Received:2023-04-06 Published:2023-10-28

摘要/Abstract

摘要：

为研究美仁牦牛抗增殖蛋白(PHB)的结构及功能,并探究其在7日龄牛和成年牛的各组织中的表达情况。以美仁牦牛肾脏组织cDNA为模板克隆美仁牦牛PHB基因序列。对基因序列进行生物信息学分析以及对蛋白质进行理化性质预测,实时荧光定量PCR(RT-qPCR)技术检测PHB基因在美仁牦牛2个年龄段8个组织中的相对表达量。结果表明,美仁牦牛PHB基因的CDS区全长819 bp,共编码272个氨基酸;同源性比对发现美仁牦牛与普通牛的同源性最高(99.1%);美仁牦牛PHB蛋白分析显示,PHB蛋白内存在1个N-糖基化潜在位点和2个O-糖基化潜在位点,其氨基酸等电点为5.55,不稳定系数(Ⅱ)为46.07,总平均疏水性为0.045,预测为不稳定疏水性蛋白质;亚细胞定位显示,PHB蛋白位于线粒体(30.4%),蛋白内共有潜在磷酸化位点15个,PHB蛋白高级结构由α-螺旋(56.25%)、无规则卷曲(22.43%)、延伸链(16.54%)和β-转角(4.78%)相互连接而成。RT-qPCR结果显示,美仁牦牛2个发育阶段的8个组织中均有PHB基因的表达。在对比2个时期的PHB基因组织表达规律后发现,7个组织的PHB基因表达量随美仁牦牛的生长发育而降低,在美仁牦牛肝脏、脾脏、肺脏、脂肪和睾丸组织中,PHB基因表达量随年龄显著降低(P<0.05)。

关键词: 美仁牦牛, PHB基因, 克隆, 生物信息学, 表达谱

Abstract:

In order to study the structure and function of PHB gene in Meiren yak,and to explore its expression in different tissues at 7-day-old and adult cattle.This study cloned the coding region of PHB gene from Meiren yak kidney tissue cDNA as a template.The gene sequence was analyzed by bioinformatics and the physical and chemical properties of the protein were predicted.The relative expression levels of PHB gene in 8 tissues of Meiren yak at two ages were detected by Real-time Fluorescence Quantitative (RT-qPCR).The results showed that the CDS region of the PHB gene of Meiren yak was 819 bp in length,encoding 272 amino acids.The results of homology analysis showed that Meiren yak had the highest homology with common cattle (99.1%).The analysis of PHB protein showed that there was one potential N-glycosylation site and two potential O-glycosylation sites in the PHB protein.Its amino acid isoelectric point was 5.55,the instability coefficient (Ⅱ) was 46.07,and the total average hydrophobicity was 0.045,which was predicted to be an unstable hydrophobic protein.Subcellular localization showed that PHB protein was located in the mitochondria (30.4%),and there were 15 potential phosphorylation sites in the protein.The high-level structure of PHB protein was interconnected by α-helix (56.25%),random coil (22.43%),extended chain (16.54%) and β-corner (4.78%).The results of RT-qPCR showed that the PHB gene was expressed in eight tissues of Meiren yak at two developmental stages.The expression of PHB gene in 7 tissues decreased with the growth and development of Meiren yak.The expression of PHB gene in liver,spleen,lung,fat and testis decreased significantly with age (P<0.05).

Key words: Meiren yak, PHB genes, Cloning, Bioinformatics, Expression profile

杨国武, 王佟, 马小勇, 扎老, 路建卫, 赵雪, 阎萍, 梁春年. 美仁牦牛PHB基因克隆及组织表达[J]. 华北农学报, 2023, 38(5): 218-226. doi: 10.7668/hbnxb.20194168.

YANG Guowu, WANG Tong, MA Xiaoyong, ZHA Lao, LU Jianwei, ZHAO Xue, YAN Ping, LIANG Chunnian. Cloning and Tissue Expression PHB Gene in Meiren Yak[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(5): 218-226. doi: 10.7668/hbnxb.20194168.

http://www.hbnxb.net/CN/Y2023/V38/I5/218

图/表 15

表1 引物序列

Tab.1 Primer sequences

基因 Genes	引物序列(5'-3') Primer sequences(5'-3')	长度/bp Length	退火温度/℃ Annealing temperature	用途 Application
PHB	F:CAGAGTGGAAGCAGGTGTGAG	1 241	58.2	PCR
	R:AAGGGTTGGTTGGAGGCTAC
PHB	F:AGTCAGCTTCCTCGCATCTTC	116	59.0	RT-qPCR
	R:AGTTCTCCAGCATCAAAGCGA
GAPDH	F:AATGAAAGGGCCATCACCATC	204	59.0	RT-qPCR
	R:GTGGTTCACGCCCATCACA

表2 生物信息学分析软件及用途

Tab.2 Bioinformatics analysis software and its application

软件 Software	功能 Function
ORF Finder	开放阅读框分析
Blast	同源性比对分析
ProtParam	蛋白质理化性质分析
ProtScale	蛋白质亲疏水性分析
SignalP 5.0	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
TargetP 2.0	蛋白亚细胞定位预测
PSORT Ⅱ	蛋白亚细胞定位预测
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 美仁牦牛PHB基因PCR扩增结果 M.DL2000 DNA Marker;1-2.PHB基因PCR扩增产物;3.空白对照。

Fig.1 PCR amplification results of PHB gene in Meiren yak M.DL2000 DNA Marker;1-2.PCR amplification products of PHB primers;3.Blank control.

图2 美仁牦牛PHB基因碱基序列对比结果

Fig.2 Comparison results of PHB gene base sequence of Meiren yak

图3 PHB基因核苷酸序列同源性对比结果

Fig.3 Nucleotide similarity analysis of PHB gene

图4 PHB基因系统进化树核苷酸置换(×100)。

Fig.4 Phylogenetic tree of PHB gene Nucleotide substitutions(×100).

表3 美仁牦牛PHB蛋白氨基酸组成

Tab.3 The amino acid composition of Meiren yak PHB protein

氨基酸 Amino acid	数量/个 Amount	含量/% Content	氨基酸 Amino acid	数量/个 Amount	含量/% Content
丙氨酸Ala(A)	32	11.8	亮氨酸Leu(L)	27	9.9
精氨酸Arg(R)	18	6.6	赖氨酸Lys(K)	12	4.4
天冬酰胺Asn(N)	7	2.6	蛋氨酸Met(M)	1	0.4
天冬氨酸Asp(D)	15	5.5	苯丙氨酸Phe(F)	13	4.8
半胱氨酸Cys(C)	1	0.4	脯氨酸Pro(P)	9	3.3
谷氨酰胺Gln(Q)	15	5.5	丝氨酸Ser(S)	17	6.2
谷氨酸Glu(E)	19	7.0	苏氨酸Thr(T)	14	5.1
甘氨酸Gly(G)	18	6.6	色氨酸Trp(W)	1	0.4
组氨酸His(H)	3	1.1	酪氨酸Tyr(Y)	4	1.5
异亮氨酸Ile(I)	21	7.7	缬氨酸Val(V)	25	9.2

表4 TargetP 2.0分析结果

Tab.4 TargetP 2.0 analysis results

基因 Gene	长度/bp Length	线粒体靶向肽/% Mitochondrial targeting peptide	分泌通路信号肽/% Secretion pathway signal peptide	其他/% Other
PHB	272	0.853	0.038	0.109

图5 美仁牦牛PHB蛋白二级结构预测图中线条从长到短依次代表α-螺旋(h)、延伸链(e)、β-转角(t)及无规则卷曲(c)。

Fig.5 The secondary structure of PHB protein in Meiren yak Lines in the figure represent,from long to short, α-helix (h),extended strand (e), β-turn (t),and random coil (c).

图6 美仁牦牛PHB蛋白质三级结构预测

Fig.6 The tertiary structure of PHB protein in Meiren yak

图7 美仁牦牛PHB蛋白疏水性预测

Fig.7 The hydrophobicity of PHB protein in Meiren yak

图8 美仁牦牛PHB蛋白序列潜在磷酸位点分析

Fig.8 The phosphorylation site of PHB protein in Meiren yak

图9 美仁牦牛PHB蛋白N-糖基化位点预测

Fig.9 The N-glycosylation sites domain of PHB protein in Meiren yak

图10 美仁牦牛PHB蛋白互作网络预测

Fig.10 The interaction network relationship of PHB protein in Meiren yak

图11 美仁牦牛PHB各组织中的相对表达量不同大写字母表示差异极显著(P<0.01),不同小写字母表示差异显著(P<0.05)。

Fig.11 The expression level of PHB in different tissues in Meiren yak Different uppercase letters indicate extremely significant difference (P<0.01),and different lowercase letters indicate significant difference (P<0.05).

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[33]	张勤文, 俞红贤, 荆海霞, 李莉, 魏青, 牛海林, 薛乾, 梁林. 基于骨骼肌线粒体超微结构研究生长期牦牛低氧适应性[J]. 畜牧兽医学报, 2013, 44(3):447-452.doi:10.3969/j.issn.0439-8114.2009.07.052.
	Zhang Q W, Yu H X, Jing H X, Li L, Wei Q, Niu H L, Xue Q, Liang L. Adaptation mechanism of developmental yak to hypoxia base on skeletal muscle mitochondria ultrastructure[J]. Acta Veterinaria et Zootechnica Sinica, 2013, 44(3):447-452.

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