九龙牦牛<i>ELOVL3</i>基因克隆、真核表达载体构建及组织表达谱分析

doi:10.7668/hbnxb.20195310

摘要/Abstract

摘要：

极长链脂肪酸延伸酶3(ELOVL3)主要参与合成极长链脂肪酸(VLCFA),在脂肪酸代谢过程中扮演重要角色。旨为克隆九龙牦牛ELOVL3基因的蛋白编码序列,构建ELOVL3基因真核表达载体,并预测分析其生物学功能,通过PCR技术扩增九龙牦牛ELOVL3基因的CDS区序列,并通过同源重组将其插入到pcDNA3.1载体中以构建重组质粒。采用限制性酶切及PCR技术验证重组质粒,结合测序结果,利用在线预测软件分析其蛋白编码序列的生物学功能。另外,针对ELOVL3基因在mRNA水平和蛋白水平的表达量,进行qPCR和Western Blot检测。结果表明:九龙牦牛ELOVL3基因的蛋白编码序列全长813 bp,共编码270个氨基酸;酶切、PCR及测序鉴定,确认成功构建pcDNA3.1-ELOVL3 真核表达载体。生物信息学分析结果显示,ELOVL3基因编码蛋白属于疏水性蛋白,共有6个跨膜结构域及27个磷酸化位点。系统发育树显示,与九龙牦牛亲缘关系最近的是普通牛,最远的是原鸡。此外,ELOVL3基因在九龙牦牛肺脏组织中表达量最高。

关键词: 牦牛, ELOVL3基因, 基因克隆, 真核表达载体构建

Abstract:

Elongation of very long chain fatty acids protein 3(ELOVL3),mainly involved in the synthesis of very long chain fatty acids(VLCFA),plays an important role in fatty acid metabolism.The objectives of this study were to clone the protein-coding sequence of ELOVL3 gene from Jiulong yak,to construct the eukaryotic expression vector of ELOVL3 gene,and to predict and analyze its biological functions.The CDS region of the ELOVL3 gene was amplified by PCR and inserted into the pcDNA3.1 vector by homologous recombination to construct a recombinant plasmid.The recombinant plasmid was validated by restriction digest and PCR,and the biological functions of its protein coding sequence were analyzed by online prediction software in combination with sequencing results.In addition,qPCR and Western Blot were performed for the expression of ELOVL3 gene at mRNA level and protein level.The results showed that the protein-coding sequence of Jiulong yak ELOVL3 gene was 813 bp in length,encoding a total of 270 amino acids.Enzymatic cleavage,PCR and sequencing identification confirmed the successful construction of the pcDNA3.1-ELOVL3 eukaryotic expression vector.Bioinformatics analysis showed that the protein encoded by the ELOVL3 gene was a hydrophobic protein,with 6 transmembrane structural domains and 27 phosphorylation sites.The phylogenetic tree showed that the closest relation to the Jiulong yak was the common cow,and the furthest was the original chicken.In addition,the ELOVL3 gene was most highly expressed in the lung tissue of the Jiulong yak.

Key words: Jiulong yak, ELOVL3 gene, Gene cloning, Eukaryotic expression vector construction

司比努尔·牙生江, 者玉琦, 钟金城, 武志娟, 柴志欣. 九龙牦牛ELOVL3基因克隆、真核表达载体构建及组织表达谱分析[J]. 华北农学报, 2025, 40(2): 201-209. doi: 10.7668/hbnxb.20195310.

SIBINUR Yashengjiang, ZHE Yuqi, ZHONG Jincheng, WU Zhijuan, CHAI Zhixin. ELOVL3 Gene Cloning,Eukaryotic Expression Vector Construction and Tissue Expression Profiling Analysis in Jiulong Yak[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(2): 201-209. doi: 10.7668/hbnxb.20195310.

http://www.hbnxb.net/CN/Y2025/V40/I2/201

图/表 14

表1 引物信息

Tab.1 Primer sequences information

基因 Genes	引物序列(5'—3') Primer sequence(5'—3')	应用 Application	扩增产物长度/bp Amplification product length
ELOVL3	F: ATGGTCACAACCGTGAATACT	扩增CDS	903
	R: CTTCCTAATCATTCCCTCAAAC
ELOVL3	F: ctagcgtttaaacttaagcttATGGTCACAACCGTGAATACT	扩增CDS	945
(同源臂)	R: ccacactggactagtggatcCCTTCCTAATCATTCCCTCAAAC
β-actin	F: GCAGGTCATCACCATCGG	qPCR检测	158
	R: CCGTGTTGGCGTAGAGGT
ELOVL3	F: ACAACCTCATTCGCCATAGC	qPCR检测	790
	R: CCCTCAGTGTCCCGAAGATA

表2 在线分析软件

Tab.2 Online analysis software

软件 Software	功能 Function	网址 Websites
ProtParam	蛋白质基本理化特性	https://web.expasy.org/protparam/
ProtScale	蛋白质的亲疏水性	https://web.expasy.org/protscale/
TMHMM-2.0	跨膜结构预测	https://services.healthtech.dtu.dk/services/TMHMM-2.0/
NetPhos-3.1	磷酸化位点预测	https://services.healthtech.dtu.dk/service.php?NetPhos-3.1
SOPMA	二级结构预测	https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html
SWISS-MODEL	三级结构预测	https://swissmodel.expasy.org/
STRING	蛋白质相互作用预测	https://string-db.org/
InterPro	功能结构域预测	https://www.ebi.ac.uk/interpro/

图1 九龙牦牛ELOVL3基因CDS区PCR扩增结果 M.2 000 bp DNA 分子质量标准;1.ELOVL3基因CDS区PCR扩增产物。

Fig.1 Results of PCR amplification of the CDS region of the ELOVL3 gene in Jiulong yak M.2 000 bp DNA Marker; 1.PCR amplification product of ELOVL3 gene CDS region.

图2 九龙牦牛ELOVL3基因真核表达载体酶切及PCR鉴定 M.2 000 bp DNA 分子量标准;1.pcDNA3.1- ELOVL3双酶切产物;2.空载体双酶切产物。

Fig.2 Digestion and PCR identification of eukaryotic expression vector of the ELOVL3 gene in Jiulong yak M.2 000 bp DNA Marker; 1.pcDNA3.1-ELOVL3 double enzyme digestion product;2.Empty vector double digestion product.

图3 ELOVL3基因在九龙牦牛不同组织中的mRNA表达丰度不同小写字母表示差异显著(P<0.05)。

Fig.3 Abundance of ELOVL3 gene mRNA expression in different tissues of Jiulong yak Values with different small letter mean significant difference(P<0.05).

图4 ELOVL3蛋白在九龙牦牛不同组织中的表达量

Fig.4 Expression of ELOVL3 protein in different tissues of Jiulong yak

图5 不同物种ELOVL3基因CDS区同源性比对

Fig.5 Homology of the CDS region of the ELOVL3 gene in different species

图6 不同物种ELOVL3基因CDS区系统发育树

Fig.6 Phylogenetic tree of CDS region of ELOVL3 gene in different species

表3 九龙牦牛ELOVL3蛋白氨基酸组成

Tab.3 Amino acid composition of Jiulong yak ELOVL3 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)	16	5.9	亮氨酸 Leu(L)	25	9.3
精氨酸 Arg(R)	9	3.3	赖氨酸 Lys(K)	15	5.6
天冬氨酰 Asn(N)	8	3.0	甲硫氨酸 Met(M)	10	3.7
天冬氨酸 Asp(D)	3	1.1	苯丙氨酸 Phe(F)	28	10.4
半胱氨酸 Cys(C)	4	1.5	脯氨酸 Pro(P)	9	3.3
谷氨酰胺 Gln(Q)	11	4.1	丝氨酸 Ser(S)	17	6.3
谷氨酸 Glu(E)	9	3.3	苏氨酸 Thr(T)	19	7.0
甘氨酸 Gly(G)	14	5.2	色氨酸 Trp(W)	9	3.3
组氨酸 His(H)	7	2.6	酪氨酸 Tyr(Y)	15	5.6
异亮氨酸 Ile(I)	22	8.1	缬氨酸 Val(V)	20	7.4

图7 九龙牦牛ELOVL3蛋白跨膜结构预测

Fig.7 Prediction of the transmembrane structure of the ELOVL3 protein in Jiulong yak

图8 九龙牦牛ELOVL3蛋白磷酸化位点预测

Fig.8 Prediction of phosphorylation sites of Jiulong yak ELOVL3 protein

图9 九龙牦牛ELOVL3蛋白二级(A)和三级(B)结构预测

Fig.9 Prediction of secondary(A)and tertiary(B) structures of ELOVL3 protein in Jiulong yak

图10 九龙牦牛ELOVL3蛋白互作预测

Fig.10 Prediction interaction of Jiulong yak ELOVL3 protein

图11 ELOVL3蛋白功能结构域预测

Fig.11 Functional domain prediction of ELOVL3 protein

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九龙牦牛ELOVL3基因克隆、真核表达载体构建及组织表达谱分析

ELOVL3 Gene Cloning,Eukaryotic Expression Vector Construction and Tissue Expression Profiling Analysis in Jiulong Yak

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