牦牛<i>DJ-1</i>基因克隆、生物信息学及组织表达分析

doi:10.7668/hbnxb.20194483

华北农学报 ›› 2024, Vol. 39 ›› Issue (2): 192-199. doi: 10.7668/hbnxb.20194483

所属专题： 畜牧；

• 畜牧・水产・兽医 • 上一篇下一篇

牦牛DJ-1基因克隆、生物信息学及组织表达分析

申金伟¹^,²^,³, 路建卫⁴, 赵雪⁵, 扎老⁴, 成述儒³, 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 甘肃农业大学,甘肃兰州 730124
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:2023-10-23 出版日期:2024-04-28
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。

成述儒(1976—),男,甘肃张掖人,教授,博士,主要从事动物遗传及生物技术研究。
作者简介:
申金伟(1999—),男,甘肃临潭人,在读硕士,主要从事动物遗传育种与繁殖研究。
基金资助:
合作市牦牛种质提升与提质增效项目; 现代肉牛牦牛产业技术体系(CARS-37); 甘肃省基础研究创新群体项目(20JR5RA580); 甘肃省科技重大专项(21ZD10NA001); 甘肃省科技重大专项(GZGG-2021-1); 中国农业科学院创新工程(25-LIHPS-01)

Cloning,Bioinformatics,and Tissue Expression Analysis of Yak DJ-1 Gene

SHEN Jinwei¹^,²^,³, LU Jianwei⁴, ZHAO Xue⁵, ZHA Lao⁴, CHENG Shuru³, LIANG Chunnian¹^,²

¹ Lanzhou Institute of Husbandry and Pharmaceutical Sciences,Chinese Academy of Agricultural Sciences, Key Laboratory of Yak Breeding Engineering Gansu Province,Lanzhou 730050,China
² Key Laboratory of Animal Genetics and Breeding on the Tibetan Plateau,Ministry of Agriculture and Rural Affairs,Lanzhou 730050, China
³ Gansu Agricultural University,Lanzhou 730124,China
⁴ Animal Husbandry and Veterinary Station, Zuogaimanma Town,Hezuo 747000,China
⁵ Agricultural and Animal Products Quality Safety Inspection Center,Hezuo 747000,China

Received:2023-10-23 Published:2024-04-28

摘要/Abstract

摘要：

为了研究牦牛DJ-1基因的结构及功能,为后续深入研究牦牛DJ-1基因的生物学功能提供了重要的理论依据。以美仁牦牛脂肪组织cDNA为模板,利用RT-PCR克隆牦牛DJ-1基因的编码区序列(CDS ),并且利用基因序列进行生物信息学分析,预测结构域及蛋白质理化性质等,再利用RT-qPCR技术进行组织表达分析。结果表明,美仁牦牛DJ-1基因的CDS区全长570 bp,共编码189个氨基酸;牦牛DJ-1结构域预测显示,在DJ-1氨基酸序列的29—168 位中有1个含有Pfam的PfpI结构域;通过对DJ-1蛋白结构和功能进行预测,结果显示,无跨膜结构且无信号肽区域,分子质量20.035 31 ku,原子总数2 870,理论等电点6.84,不稳定系数28.37,表示为稳定的蛋白质;脂肪系数101.11,总平均亲水系数-0.004,为亲水蛋白,共有11个磷酸化位点;同时,亚细胞定位预测结果表明,美仁牦牛DJ-1蛋白主要分布于细胞质和线粒体中,系统进化树表明,美仁牦牛与野牦牛和欧洲牛亲缘关系最近,与北极狐和宽吻海牛亲缘关系最远;RT-qPCR结果表明,在成年美仁牦牛的心脏、肝脏、脾脏、肺脏、肌肉、脂肪和睾丸组织中均有所表达,在心脏组织和肌肉组织中表达水平最高,在肝脏和睾丸组织中表达水平最低。

关键词: 美仁牦牛, DJ-1基因, 生物信息学分析, 系统进化

Abstract:

In order to study the structure and function of DJ-1 gene in yaks,this experiment used Meiren yak adipose tissue cDNA as a template,cloned the coding sequence (CDS) of yak DJ-1 gene using RT-PCR,and performed bioinformatics analysis,including domain prediction and protein physicochemical properties prediction of the gene sequence.Then,tissue expression analysis was performed using RT-qPCR.The results showed that the CDS region of the DJ-1 gene in Meiren yak had a total length of 570 bp and encoded 189 amino acids;the prediction of the DJ-1 domain in yaks showed that there was one PfpI domain containing the Pfam in positions 29—168 of the DJ-1 amino acid sequence.By predicting structure and function of the DJ-1 protein,the results showed no transmembrane structure and no signal peptide region;molecular weight was 20.035 31 ku,total number of atoms was 2 870,theoretical isoelectric point was 6.84,instability coefficient was 28.37,and expressed as a stable protein;the fat coefficient was 101.11,with a total average hydrophilicity coefficient of -0.004.It was a hydrophilic protein with a total of 11 phosphorylation sites.Meanwhile,the results of subcellular localization prediction indicated that the DJ-1 protein of Meiren yak was mainly distributed in the cytoplasm and mitochondria.The phylogenetic tree showed that Meiren yak had the closest genetic relationship with wild yaks and European cattle,and the farthest genetic relationship with Arctic foxes and wide snouted manatees;the RT-qPCR results showed that it was expressed in the heart,liver,spleen,lungs,muscle,fat,and testicular tissues of adult Meiren yaks,with the highest expression level in the heart and muscle tissues,and the lowest expression level in the liver and testicular tissues.

Key words: Meiren yak, DJ-1 gene, Bioinformatics analysis, System evolution

申金伟, 路建卫, 赵雪, 扎老, 成述儒, 梁春年. 牦牛DJ-1基因克隆、生物信息学及组织表达分析[J]. 华北农学报, 2024, 39(2): 192-199. doi: 10.7668/hbnxb.20194483.

SHEN Jinwei, LU Jianwei, ZHAO Xue, ZHA Lao, CHENG Shuru, LIANG Chunnian. Cloning,Bioinformatics,and Tissue Expression Analysis of Yak DJ-1 Gene[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(2): 192-199. doi: 10.7668/hbnxb.20194483.

http://www.hbnxb.net/CN/Y2024/V39/I2/192

图/表 17

表1 引物信息

Tab.1 Primer information

基因 Gene	引物序列(5'—3') Primer sequences(5'—3')	片段大小/bp Fragment size	退火温度/℃ Tm	用途 Usage
DJ-1	F:GGCGAGGTTCAGGTCTTCTA	714	58	DJ-1 CDS扩增
	R:CACAACGAGGCTGGATTACAA
DJ-1	F: CCTCATCGCTGCCATCTGT	140	55	实时荧光定量PCR
	R: CCACACGGTTCTCGGAGTAG
GADPH	F:AATGAAAGGGCCATCACCATC	204	53	实时荧光定量PCR
	R:GTGGTTCACGCCCATCACA

表2 生物信息学分析软件及用途

Tab.2 Bioinformatics analysis software and its applications

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
BLAST	https://blast.ncbi.nlm.nih.gov/Blast.cgi	同源性比对分析
ProtParam	https://web.expasy.org/protparam/	蛋白质理化性质分析
ProtScale	https://web.expasy.org/protscale/	蛋白质亲疏水性分析
SignalP 4.1	http://www.cbs.dtu.dk/services/SignalP-4.1/	蛋白质信号肽分析
TMHMM	http://www.cbs.dtu.dk/services/TMHMM/	蛋白质跨膜结构预测
SMART	http://smart.embl-heidelberg.de/	蛋白质结构域预测
PSORT Ⅱ	https://www.genscript.com/psort.html	蛋白亚细胞定位预测
SOPMA	https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.plpage=npsa_sopma.html	蛋白质二级结构预测
SWISS-MODEL	https://swissmodel.expasy.org/interactive	蛋白质三级结构预测
STRING 10.5	https://www.string-db.org/	蛋白互作网络预测

图1 DJ-1基因扩增产物

Fig.1 DJ-1 gene amplification product

图2 美仁牦牛DJ-1基因开放阅读框

Fig.2 Meiren yak DJ-1 gene open reading frame

图3 美仁牦牛DJ-1基因氨基酸序列

Fig.3 Meiren yak DJ-1 gene amino acid sequence

表3 美仁牦牛DJ-1蛋白氨基酸组成成分

Tab.3 Amino acid composition of DJ-1 protein in Meiren yak

序号 Serial number	氨基酸 Amino acid	数量/个 Quantity	含量/% Content	序号 Serial number	氨基酸 Amino acid	数量/个 Quantity	含量/% Content
1	丙氨酸Ala(A)	21	11.1	11	亮氨酸Leu(L)	18	9.5
2	精氨酸Arg(R)	7	3.7	12	赖氨酸Lys(K)	18	9.5
3	天冬酰胺Asn(N)	4	2.1	13	蛋氨酸Met(M)	5	2.6
4	天冬氨酸Asp(D)	10	5.3	14	苯丙氨酸Phe(F)	3	1.6
5	半胱氨酸Cys(C)	3	1.6	15	脯氨酸Pro(P)	9	4.8
6	谷氨酰胺Gln(Q)	4	2.1	16	丝氨酸Ser(S)	11	5.8
7	谷氨酸Glu(E)	15	7.9	17	苏氨酸Thr(T)	7	3.7
8	甘氨酸Gly(G)	17	9.0	18	色氨酸Trp(W)	0	0.0
9	组氨酸His(H)	3	1.6	19	酪氨酸Tyr(Y)	3	1.6
10	异亮氨酸Ile(I)	10	5.3	20	缬氨酸Val(V)	21	11.1

图4 美仁牦牛DJ-1蛋白疏水性预测

Fig.4 Prediction of hydrophobicity of DJ-1 protein in Meiren yak

图5 美仁牦牛DJ-1蛋白结构域预测

Fig.5 Prediction of DJ-1 protein domain in Meiren yak

图6 美仁牦牛DJ-1蛋白磷酸化位点分析

Fig.6 Analysis of phosphorylation sites of DJ-1 protein in Meiren yak

图7 美仁牦牛DJ-1蛋白二级结构预测 h.α-螺旋;e.延伸链;t.β-转角;c.无规则卷曲。

Fig.7 Prediction of the secondary structure of DJ-1 protein in Meiren yak h.α-helix;e.Extended strand;t.β-turn;c.Random coil.

图8 美仁牦牛DJ-1蛋白三级结构预测

Fig.8 Prediction of the tertiary structure of DJ-1 protein in Meiren yak

图9 美仁牦牛DJ-1基因编码蛋白跨膜结构预测

Fig.9 Prediction of transmembrane structure of DJ-1 gene encoded protein in Meiren yak

图10 美仁牦牛DJ-1基因编码蛋白信号肽预测

Fig.10 Prediction of protein signal peptides encoded by DJ-1 gene in Meiren yak

图11 美仁牦牛DJ-1基因翻译蛋白互作网络分析

Fig.11 Analysis of translation protein interaction network of DJ-1 gene in Meiren yak

图12 美仁牦牛DJ-1基因与其余物种基因构建的系统进化树

Fig.12 Phylogenetic tree constructed by the DJ-1 gene of Meiren yak and other species

图13 DJ-1基因核苷酸序列同源性比对

Fig.13 Nucleotide sequence homology alignment of DJ-1 gene

图14 不同组织DJ-1基因的mRNA表达水平不同大写字母表示差异极显著(P<0.01)。

Fig.14 The mRNA expression level of DJ-1 gene Different capital indicate significant differences in different tissues (P<0.01).

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牦牛DJ-1基因克隆、生物信息学及组织表达分析

Cloning,Bioinformatics,and Tissue Expression Analysis of Yak DJ-1 Gene

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