麦洼牦牛<i>MFSD4A</i>基因克隆及组织表达分析

doi:10.7668/hbnxb.20194563

摘要/Abstract

摘要：

旨在克隆麦洼牦牛MFSD4A基因序列并阐明其在不同组织中的表达模式,分析MFSD4A互作蛋白mRNA表达水平及其相关性,确定MFSD4A蛋白的亚细胞定位,为后续MFSD4A在牦牛睾丸生长发育调控研究中提供理论依据。以4头健康麦洼牦牛的心、肝、脾、肺、肾、肌肉、脂肪、臀肌、背部肌肉、睾丸、结肠组织作试验材料,用 RT-PCR技术克隆麦洼牦牛MFSD4A 基因CDS序列并对其进行生物信息学分析;利用RT-qPCR技术检测MFSD4A基因在心、肝、脾、肺、肾、臀肌、背部肌肉、脂肪、睾丸、淋巴等10个组织中的表达及与其互作蛋白的相关性;构建pEGFP-MFSD4A融合质粒转染牛肾细胞系(MDBK)探究MFSD4A蛋白的亚细胞定位情况。结果表明,麦洼牦牛MFSD4A基因 CDS 区全长为1 530 bp,编码509个氨基酸,理论等电点(pI)为8.66,具有12个跨膜结构域,属于碱性跨膜蛋白;RT-qPCR结果显示,MFSD4A mRMA在麦洼牦牛各组织中差异表达,且在睾丸组织中表达量最高,与MFSD9、CBY1、INHBA、UNC93A、SVOP、ID1在睾丸组织中的表达量呈极显著正相关,推测MFSD4A基因可能与牦牛睾丸的生长发育调控有关;构建 pEGFP-MFSD4A 融合质粒转染牛肾细胞(MDBK)后的荧光定位显示该蛋白主要定位于细胞核。

关键词: 牦牛, MFSD4A, 基因克隆, 互作蛋白分析, 亚细胞定位

Abstract:

This study aims to obtain the MFSD4A gene sequence in Maiwa yak and elucidate its expression patterns in different tissues,analyze the mRNA expression levels of the interacting proteins of MFSD4A,and determine the subcellular localization of the MFSD4A protein,and provide a theoretical basis for further research on regulating MFSD4A in the growth and development of yak testis.RT-PCR was used to obtain the MFSD4A gene CDS sequence in Maiwa yak and perform bioinformatics analysis.RT-qPCR was used to detect the expression differences of the MFSD4A gene in 10 tissues,including heart,liver,spleen,lung,kidney,gluteal muscle,back muscle,fat,testis,and lymph,and the correlation between the interacting proteins and MFSD4A.pEGFP-MFSD4A fusion plasmid was transfected into Madin-Darby bovine kidney cells(MDBK)to investigate the subcellular localization of the MFSD4A protein.The results indicated that the entire length of the MFSD4A gene CDS region in the macaque yak was 1 530 bp,encoding 509 amino acids with a theoretical isoelectric point(PI)of 8.66.It had 12 transmembrane domains and belonged to the alkaline transmembrane protein.RT-qPCR results showed differential expression of MFSD4A mRNA in various tissues of the macaque yak,with the highest expression in the testis.It was also highly positively correlated with the expression levels of MFSD9,CBY1,INHBA,UNC93A,SVOP,and ID1 in the testis,suggesting that the MFSD4A gene might be related to the regulation of growth and development of yak testis.Fluorescence localization of the pEGFP-MFSD4A fusion vector transfected into Madin-Darby bovine kidney cells(MDBK)showed that the protein was mainly located in the nucleus.

Key words: Yak, MFSD4A, Gene cloning, Interaction protein analysis, Subcellular localization

胡莲, 刘益丽, 赵迪, 王泽宁, 江明锋. 麦洼牦牛MFSD4A基因克隆及组织表达分析[J]. 华北农学报, 2024, 39(2): 174-183. doi: 10.7668/hbnxb.20194563.

HU Lian, LIU Yili, ZHAO Di, WANG Zening, JIANG Mingfeng. Cloning and Tissue Expression Analysis of MFSD4A Gene in Maiwa Yak[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(2): 174-183. doi: 10.7668/hbnxb.20194563.

http://www.hbnxb.net/CN/Y2024/V39/I2/174

图/表 12

参考文献 33

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分析工具 Analysis tool	网址 URL	功能 Function
ORF Finder	https://www.ncbi.nlm.nih.gov/orffinder/	开放阅读框预测
SNAP2	https://rostlab.org/services/snap2web/	序列变异的功能效应预测
ExPASy-ProtParam	https://web.expasy.org/protparam/	蛋白质理化性质分析
SOPMA	https://npsa-prabi.ibcp.fr/NPSA/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
SignalP-4.1	http://www.cbs.dtu.dk/services/SignalP-4.1/	信号肽分析
TMHMM-2.0	https://services.healthtech.dtu.dk/service.php?TMHMM-2.0	蛋白质跨膜结构分析
NetPhos-3.1	https://services.healthtech.dtu.dk/services/SignalP-4.1/	蛋白质磷酸位点预测
SMART	http://smart.embl-heidelberg.de/	蛋白质结构与分析
STRING	https://string db.org/	蛋白质相互作用分析
DNAMAN 6.0	https://www.lynnon.com/dnaman.html/	物种间氨基酸序列比对
MEGA 7.0	https://www.megasoftware.net/	系统进化树构建
Omicshare	https://www.genedenovo.com/	GO富集分析

分析工具 Analysis tool	网址 URL	功能 Function
ORF Finder	https://www.ncbi.nlm.nih.gov/orffinder/	开放阅读框预测
SNAP2	https://rostlab.org/services/snap2web/	序列变异的功能效应预测
ExPASy-ProtParam	https://web.expasy.org/protparam/	蛋白质理化性质分析
SOPMA	https://npsa-prabi.ibcp.fr/NPSA/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
SignalP-4.1	http://www.cbs.dtu.dk/services/SignalP-4.1/	信号肽分析
TMHMM-2.0	https://services.healthtech.dtu.dk/service.php?TMHMM-2.0	蛋白质跨膜结构分析
NetPhos-3.1	https://services.healthtech.dtu.dk/services/SignalP-4.1/	蛋白质磷酸位点预测
SMART	http://smart.embl-heidelberg.de/	蛋白质结构与分析
STRING	https://string db.org/	蛋白质相互作用分析
DNAMAN 6.0	https://www.lynnon.com/dnaman.html/	物种间氨基酸序列比对
MEGA 7.0	https://www.megasoftware.net/	系统进化树构建
Omicshare	https://www.genedenovo.com/	GO富集分析

基因 Gene		引物序列(5'—3') Primer sequence(5'—3')	退火温度/℃ Annealing temperature	扩增产物长度/bp Amplified products size	用途 Usage
MFSD4A		F:TGCTGGTCGGCTCGATATTC	60	171	RT-qPCR
		R:GGCTCTCCATTCCAAGTGCT
GAPDH		F:TGTTGTGGATCTGACCTGCC	60	135	RT-qPCR
		R:AAGTCGCAGGAGACAACCTG
SCNN1A		F: CCACCACCCGATGTATGGAA	60	101	RT-qPCR
		R: TCAGGGACAGACCGTTGTTG
CAMSAP1		F: GCTCCTTTAGTGTTGAAGCCAA	60	95	RT-qPCR
		R: TCCCTGGGCTGGACAAAATC
SVOPL		F:ATTGAGACCAAAGGACGGGC	60	111	RT-qPCR
		R:TAAATGTGCCACAGCTCTTTGT
MFSD9		F: ACCTGGTAGGCTTCCTGGATT	60	107	RT-qPCR
		R: GCCTACTATCCCAGCAACCG
CBY1	F: TTGGAAGTACGTTTAGTCCGAAGA		60	115	RT-qPCR
	R: GTTCCGTAGTCAAGGCCCAGT
INHBA	F: AGAGGACGACATCGGACGGA		60	133	RT-qPCR
	R: CAGGTCACTGCCTTCTTTGGA
UNC93A	F: GCGCAGAAGACGGGACAAGT		60	117	RT-qPCR
	R: GCCGAACACGAGAGACGAGA
MFSD10	F: AGGCCATCTTGCTGCTCATC		60	98	RT-qPCR
	R: AGACGCCCAGGAGTAGAGG
SVOP	F: CTCCTCTTTGCGGTCCTGTG		60	166	RT-qPCR
	R: TGCCTCGGTCTTCCTGTCTG
ID1	F: TACTCTACGACATGAACGGCTGC		60	132	RT-qPCR
	R: GCTCCAACTCCAGATCCCAGA

基因 Gene		引物序列(5'—3') Primer sequence(5'—3')	退火温度/℃ Annealing temperature	扩增产物长度/bp Amplified products size	用途 Usage
MFSD4A		F:TGCTGGTCGGCTCGATATTC	60	171	RT-qPCR
		R:GGCTCTCCATTCCAAGTGCT
GAPDH		F:TGTTGTGGATCTGACCTGCC	60	135	RT-qPCR
		R:AAGTCGCAGGAGACAACCTG
SCNN1A		F: CCACCACCCGATGTATGGAA	60	101	RT-qPCR
		R: TCAGGGACAGACCGTTGTTG
CAMSAP1		F: GCTCCTTTAGTGTTGAAGCCAA	60	95	RT-qPCR
		R: TCCCTGGGCTGGACAAAATC
SVOPL		F:ATTGAGACCAAAGGACGGGC	60	111	RT-qPCR
		R:TAAATGTGCCACAGCTCTTTGT
MFSD9		F: ACCTGGTAGGCTTCCTGGATT	60	107	RT-qPCR
		R: GCCTACTATCCCAGCAACCG
CBY1	F: TTGGAAGTACGTTTAGTCCGAAGA		60	115	RT-qPCR
	R: GTTCCGTAGTCAAGGCCCAGT
INHBA	F: AGAGGACGACATCGGACGGA		60	133	RT-qPCR
	R: CAGGTCACTGCCTTCTTTGGA
UNC93A	F: GCGCAGAAGACGGGACAAGT		60	117	RT-qPCR
	R: GCCGAACACGAGAGACGAGA
MFSD10	F: AGGCCATCTTGCTGCTCATC		60	98	RT-qPCR
	R: AGACGCCCAGGAGTAGAGG
SVOP	F: CTCCTCTTTGCGGTCCTGTG		60	166	RT-qPCR
	R: TGCCTCGGTCTTCCTGTCTG
ID1	F: TACTCTACGACATGAACGGCTGC		60	132	RT-qPCR
	R: GCTCCAACTCCAGATCCCAGA

基因 Gene	MFSD4A	SCNN1A	CAMSAP1	SVOPL	MFSD9	CBY1	INHBA	UNC93A	MFSD10	SVOP	ID1
MFSD4A	1.000
SCNN1A	0.277	1.000
CAMSAP1	0.591^*	0.295	1.000
SVOPL	0.583^*	-0.285	0.695^*	1.000
MFSD9	0.840^**	0.401	0.922^**	0.661^*	1.000
CBY1	0.798^**	0.017	0.024	0.370	0.360	1.000
INHBA	0.739^**	0.163	0.953^**	0.840^**	0.942^**	0.278	1.000
UNC93A	0.926^**	0.024	0.431	0.710^**	0.678^*	0.896	0.659^*	1.000
MFSD10	0.695^*	0.003	0.885^**	0.937^**	0.862^**	0.314	0.973^**	0.699^*	1.000
SVOP	0.863^**	-0.013	0.500	0.805^**	0.687^*	0.811	0.719^**	0.966^**	0.773^**	1.000
ID1	0.711^**	0.321	0.974^**	0.720^**	0.957^**	0.184	0.960^**	0.563	0.902^**	0.596^*	1.000

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麦洼牦牛MFSD4A基因克隆及组织表达分析

Cloning and Tissue Expression Analysis of MFSD4A Gene in Maiwa Yak

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麦洼牦牛MFSD4A基因克隆及组织表达分析

Cloning and Tissue Expression Analysis of MFSD4A Gene in Maiwa Yak

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