合作猪<i>StAR</i>基因克隆、组织表达及生物信息学分析

doi:10.7668/hbnxb.20193960

摘要/Abstract

摘要：

旨在对合作猪StAR基因CDS区进行克隆及生物信息学分析,采用RT-qPCR方法检测StAR基因在不同组织中的表达量,以揭示其功能。结果发现,合作猪StAR基因CDS区长858 bp,编码285个氨基酸,与猪参考序列相比,第572位点处的A突变为G,导致第191位的赖氨酸突变成精氨酸,为错义突变,第791位点处插入1个碱基C,后续的20个氨基酸发生改变,导致移码突变;蛋白分子量90.95 ku,理论等电点8.87;消光系数33 835,不稳定系数41.49;含1个N-糖基化位点,二级结构主要由α螺旋和无规则卷曲构成,二级结构与三级结构基本一致;合作猪与猪、绵羊和黄牛StAR基因核苷酸序列相似性分别为99.77%,90.45%,91.78%;进化树结果显示,合作猪与猪亲缘关系最近,表明StAR基因编码序列具有种属特异性;StAR蛋白是不稳定的亲水性蛋白,无信号肽和跨膜区;StAR基因在脾、肾、肺等组织中均有表达,睾丸表达量较高,表明可能与合作猪睾丸发育有关。

关键词: 合作猪, StAR基因, 生物信息学, 组织表达

Abstract:

The purpose of this paper was to clone the CDS region of StAR gene,perform bioinformatics and detect StAR gene expression level among different tissues using RT-qPCR in order to revel its function.The results showed that the CDS region of Hezuo pig StAR gene was 858 bp in length,encoding 285 amino acids.Compared to the reference sequence of pig,the A at the 572nd point mutation to G(leading to lysine mutation to arginine at the 191st point)was missense mutation and insertion of a base C at the 791st point(causing 20 amino acids mutation)was frame shift mutation,respectively.Molecular mass was about 90.95 ku and theoretical isoelectric point was 8.87;extinction coefficient was 33 835 and unstable coefficient was 41.49.Additionally,it contained one N-sugar-based site and secondary structure was mainly composed of α spiral and irregular curling.The secondary structure was similar to the tertiary structure.Lastly,the similarity of StAR nucleic acid sequences among Hezuo pig and Sus scrofa, Ovis aries, Bos taurus was 99.77%, 90.45% and 91.78%,respectively.The phylogenetic tree result showed that Hezuo pig was closed to pig.StAR gene expressed in these tissues,such as spleen,kidney,lung.Among these tissues,testis have higher expression,indicating that it may be related to the development testis in Hezuo pig.

Key words: Hezuo pig, StAR gene, Bioinformatics, Tissue expression

闫尊强, 计亚楠, 王鹏飞, 张博, 石海霞, 滚双宝. 合作猪StAR基因克隆、组织表达及生物信息学分析[J]. 华北农学报, 2023, 38(4): 190-196. doi: 10.7668/hbnxb.20193960.

YAN Zunqiang, JI Yanan, WANG Pengfei, ZHANG Bo, SHI Haixia, GUN Shuangbao. Cloning,Tissue Expression and Bioinformatics Analysis of StAR Gene from Hezuo Pig[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(4): 190-196. doi: 10.7668/hbnxb.20193960.

http://www.hbnxb.net/CN/Y2023/V38/I4/190

图/表 15

表1 引物信息

Tab.1 Primers information

基因名 Gene name	转录本号 Transcript No.	引物序列(5'—3') Primer sequences (5'—3')	长度/bp Length	用途 Purpose
StAR	NM_213755.2	F1:CCTCCGCTACCAGGAAACAATG	858	PCR扩增
		R1:TGGCGGGCTGAGCTTTAACA
		F2:TTCGACGTCGGAGCTCTCT	180	定量
		R2:CTTTACTCAGCACCTCGTCCC
GAPDH	NM_001206359.1	F:AGCAATGCCTCCTGTACCAC	210	定量
		R:AAGCAGGGATGATGTTCTGG

表2 生物信息学分析工具

Tab.2 Bioinformatics analysis tools

软件 Software	网址 Website	功能用途 Function
Blast	https://blast.ncbi.nlm.nih.gov/Blast.cgi	序列比对
ProtParam	https://www.expasy.org/resources/protparam	蛋白质理化性质分析
ProtScale	https://web.expasy.org/protscale/	蛋白质疏水性分析
TMHMM	https://services.healthtech.dtu.dk/service.php?TMHMM-2.0/	跨膜区分析
SignalP 5.0	http://www.cbs.dtu.dk/services/SignalP/	信号肽分析
SMART	http://smart.embl.de/	结构域分析
PSORT Ⅱ	https://psort.hgc.jp/form2.html	亚细胞定位分析
SOPMA	https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html	二级结构预测
Swiss-Model	http://swissmodel.expasy.org/	三级结构预测
NetNGlyc 1.0	http://www.cbs.dtu.dk/services/NetNGlyc/	N糖基化位点预测
STRING	https://cn.string-db.org/	蛋白相互作用分析

图1 合作猪StAR基因PCR扩增

Fig.1 PCR amplification of Hezuo pig StAR gene

图2 合作猪StAR基因测序结果分析

Fig.2 Analysis of sequencing results of Hezuo pig StAR gene

图3 合作猪StAR蛋白氨基酸序列比对

Fig.3 Alignment of amino acid sequences of Hezuo pig StAR protein

表3 合作猪StAR基因与其他物种序列比对

Tab.3 Sequence comparison of Hezuo pig StAR gene with other species %

物种 Species	核苷酸序列相似性 Nucleic acid sequence similarity	氨基酸序列相似性 Amino acid sequence similarity
人 Homo sapiens	87.76	89.32
绵羊 Ovis aries	90.45	90.04
狗 Canis lupus	88.36	89.68
黑猩猩 Pan troglodytes	88.31	87.38
家鼠 Mus musculus	—	86.83
马 Equus caballus	88.76	89.68
猕猴 Macaca mulatta	87.19	87.90
黄牛 Bos taurus	91.78	90.04
猪 Sus scrofa	99.77	90.12
鸡 Gallus gallus	—	69.37

图4 StAR基因系统进化树分析

Fig.4 Phylogenetic tree analysis of StAR gene

图5 合作猪StAR蛋白的氨基酸组成及频率

Fig.5 Amino acid composition and frequency Hezuo pig StAR protein

图6 合作猪StAR蛋白二级结构(A)及三级结构(B) h.α-螺旋;t.β-转角;c.无规则卷曲;e.延伸链。

Fig.6 Secondary structure(A)and tertiary structure(B)of StAR protein in Hezuo pig h.Alpha-helix;t.Beta-angle;c.Random crimp;e.Extension chain

图7 合作猪StAR蛋白的疏水性分析

Fig.7 Hezuo pig StAR protein hydrophobicity analysis

图8 合作猪StAR蛋白的跨膜区域(A)及信号肽(B)

Fig.8 Transmembrane region(A)and signal peptide(B)of Hezuo pig StAR protein

图9 合作猪StAR蛋白保守结构域预测

Fig.9 Prediction of conserve structure domain of Hezuo pig StAR protein

图10 合作猪StAR蛋白N-糖基化位点及亚细胞定位

Fig.10 Hezuo pig StAR protein N-glycosylation site and subcellular localization

图11 合作猪StAR蛋白网络互作分析图

Fig.11 StAR protein network interaction analysis in Hezuo pig

图12 StAR基因在合作猪不同组织中的表达量不同小写字母表示差异显著(P<0.05);不同大写字母表示差异极显著(P<0.01)。

Fig.12 StAR gene expression level in Hezuo pig different tissues Different lowercase letters indicated significant difference(P<0.05);different uppercase letters indicated extremely significant difference(P<0.01).

参考文献 26

[1]	乌日罕, 哈斯高娃, 陈小辛, 张睿, 娜哈雅, 娜仁花. 类固醇激素合成急性调节蛋白(StAR)的研究进展[J]. 当代畜禽养殖业, 2021(4):20-26.doi:10.3969/j.issn.1005-5959.2021.04.007. doi: 10.3969/j.issn.1005-5959.2021.04.007
	Wu R H, Ha S, Chen X X, Zhang R, Na H Y, Na R H. Research progress in steroid hormone synthesis of acute regulatory protein(StAR)[J]. Modern Livestock and Poultry Breeding Industry, 2021(4):20-26.
[2]	李娟, 栾新红. 类固醇合成急性调节蛋白的生物学特性及作用研究概况[J]. 动物医学进展, 2016, 37(1):110-113.doi:10.3969/j.issn.1007-5038.2016.01.025. doi: 10.3969/j.issn.1007-5038.2016.01.025
	Li J, Luan X H. Induction to biological characteristics and action mechanisms of steroidogenic acute regulatory protein[J]. Progress in Veterinary Medicine, 2016, 37(1):110-113.
[3]	王草叶, 王玉邦. 类固醇激素合成急性调节蛋白研究进展[J]. 中华男科学杂志, 2006, 12(8):733-736.doi:10.3969/j.issn.1009-3591.2006.08.018. doi: 10.3969/j.issn.1009-3591.2006.08.018
	Wang C Y, Wang Y B. Advances in the study of steroidogenic acute regulatory protein[J]. National Journal of Andrology, 2006, 12(8):733-736.
[4]	Bose H S, Whittal R M, Marshall B, Rajapaksha M, Wang N P, Bose M, Perry E W, Zhao Z Q, Miller W L. A novel mitochondrial complex of aldosterone synthase,steroidogenic acute regulatory protein,and Tom 22 synthesizes aldosterone in the rat heart[J]. The Journal of Pharmacology and Experimental Therapeutics, 2021, 377(1):108-120.doi:10.1124/jpet.120.000365. doi: 10.1124/jpet.120.000365 URL
[5]	刘宇, 邬建飞, 李恒, 荆天, 卢建远, 字向东. 牦牛StAR基因克隆及其生物信息学与组织表达特性的研究[J]. 畜牧兽医学报, 2021, 52(9):2452-2463.doi:10.11843/j.issn.0366-6964.2021.09.008. doi: 10.11843/j.issn.0366-6964.2021.09.008
	Liu Y, Wu J F, Li H, Jing T, Lu J Y, Zi X D. Cloning and analysis of bioinformatics and tissue expression characteristics of yak StAR gene[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(9):2452-2463.
[6]	Lavigne P, Najmanivich R, LeHoux J G. Mammalian StAR-related lipid transfer(START)domains with specificity for cholesterol:Structural conservation and mechanism of reversible binding[J]. Sub-cellular Biochemistry, 2010, 51: 425-437.doi:10.1007/978-90-481-8622-8-15. doi: 10.1007/978-90-481-8622-8_15 pmid: 20213553
[7]	Christenson L K, Strauss J F. Steroidogenic acute regulatory protein:An update on its regulation and mechanism of action[J]. Archives of Medical Research, 2001, 32(6):576-586.doi:10.1016/S0188-4409(01)00338-1. doi: 10.1016/S0188-4409(01)00338-1 pmid: 11750733
[8]	Tuckey R C, Headlam M J, Bose H S, Miller W L. Transfer of cholesterol between phospholipid vesicles mediated by the steroidogenic acute regulatory protein(StAR)[J]. Journal of Biological Chemistry, 2002, 277(49):47123-47128.doi:10.1074/jbc.M206965200. doi: 10.1074/jbc.M206965200 URL
[9]	Lin D, Sugawara T, Strauss J F, Clark B J, Stocco D M, Saenger P, Rogol A, Miller W L. Role of steroidogenic acute regulatory protein in adrenal and gonadal steroidogenesis[J]. Science, 1995, 267(5205):1828-1831.doi:10.1126/science.7892608. doi: 10.1126/science.7892608 pmid: 7892608
[10]	Kang E, Kim Y M, Kim G H, Lee B H, Yoo H W, Choi J H. Mutation spectrum of STAR and the founder effect of p.Q258* in Korean patients with congenital lipoid adrenal hyperplasia[J]. Molecular Medicine, 2017, 23(1):149-154.doi:10.2119/molmed.2017.00023. doi: 10.2119/molmed.2017.00023
[11]	Leers-sucheta S, Stocco D M, Azhar S. Down-regulation of steroidogenic acute regulatory(StAR)protein in rat Leydig cells:implications for regulation of testosterone production during aging[J]. Mechanisms of Ageing and Development, 1999, 107(2):197-203.doi:10.1016/S0047-6374(98)00149-3. doi: 10.1016/S0047-6374(98)00149-3 pmid: 10220047
[12]	Stocco D M. StAR protein and the regulation of steroid hormone biosynthesis[J]. Annual Review of Physiology, 2001, 63:193-213.doi:10.1146/annurev.physiol.63.1.193. doi: 10.1146/annurev.physiol.63.1.193 pmid: 11181954
[13]	Hasegawa T, Zhao L P, Caron K M, Majdic G, Suzuki T, Shizawa S, Sasano H, Parker K L. Developmental roles of the steroidogenic acute regulatory protein(StAR)as revealed by StAR knockout mice[J]. Molecular Endocrinology, 2000, 14(9):1462-1471.doi:10.1210/me.14.9.1462. doi: 10.1210/mend.14.9.0515 pmid: 10976923
[14]	刘孟洲, 王玉涛. 合作猪种质特性的研究[J]. 中国畜牧杂志, 2006, 42(13):6-8.doi:10.3969/j.issn.0258-7033.2006.13.003. doi: 10.3969/j.issn.0258-7033.2006.13.003
	Liu M Z, Wang Y T. Study on breed characteristic of Hezuo pig[J]. Chinese Journal of Animal Science, 2006, 42(13):6-8.
[15]	陈国顺, 滚双宝. 合作猪种质资源调查研究[J]. 猪业科学, 2013, 30(9):124-127.doi:10.3969/j.issn.1673-5358.2013.09.033. doi: 10.3969/j.issn.1673-5358.2013.09.033
	Chen G S, Gun S B. Investigation and study on cooperative pig germplasm resources[J]. Swine Industry Science, 2013, 30(9):124-127.
[16]	Zhang B, Yan Z Q, Gao Y, Li J Y, Wang Z K, Wang P F, Yang Q L, Huang X Y, Gun S B. Integrated analysis of miRNA and mRNA expression profiles in testes of Landrace and Hezuo boars[J]. Frontiers in Veterinary Science, 2022, 9:942669.doi:10.3389/fvets.2022.942669. doi: 10.3389/fvets.2022.942669 URL
[17]	Zhang B, Yan Z Q, Wang P F, Yang Q L, Huang X Y, Shi H X, Tang Y R, Ji Y N, Zhang J L, Gun S B. Identification and characterization of lncRNA and mRNA in testes of Landrace and Hezuo boars[J]. Animals, 2021, 11(8):2263.doi:10.3390/ani11082263. doi: 10.3390/ani11082263 URL
[18]	Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method[J]. Methods, 2001, 25(4):402-408.doi:10.1006/meth.2001.1262. doi: 10.1006/meth.2001.1262 pmid: 11846609
[19]	Manna P R, Stetson C L, Slominski A T, Pruitt K. Role of the steroidogenic acute regulatory protein in health and disease[J]. Endocrine, 2016, 51(1):7-21.doi:10.1007/s12020-015-0715-6. doi: 10.1007/s12020-015-0715-6 pmid: 26271515
[20]	Miller W L. Steroidogenic acute regulatory protein(StAR),a novel mitochondrial cholesterol transporter[J]. Biochimica et BiophysicaActa-Molecular and Cell Biology of Lipids, 2007, 1771(6):663-676.doi:10.1016/j.bbalip.2007.02.012. doi: 10.1016/j.bbalip.2007.02.012
[21]	Chen H, Zhang Q R, Chen R M, Yuan X, Lin X Q, Yang X H, Zhang Y. Lipoid congenital adrenal hyperplasia due to steroid acute regulatory protein(STAR)variants in three Chinese patients[J]. The Journal of Steroid Biochemistry and Molecular Biology, 2020, 200:105635.doi:10.1016/j.jsbmb.2020.105635. doi: 10.1016/j.jsbmb.2020.105635 URL
[22]	张育辉, 李霞. 类固醇激素合成急性调节蛋白的研究进展[J]. 陕西师范大学学报(自然科学版), 2009, 37(3):85-89.doi:10.19556/j.0258-7033.20210818-07. doi: 10.19556/j.0258-7033.20210818-07
	Zhang Y H, Li X. Research progress in steroidogenic acute regulatory protein[J]. Journal of Shaanxi Normal University (Natural Science Edition), 2009, 37(3):85-89.
[23]	Strickland J, McIlmoil S, Williams B J, Seager D C, Porter J P, Judd A M. Interleukin-6 increases the expression of key proteins associated with steroidogenesis in human NCI-H295R adrenocortical cells[J]. Steroids, 2017, 119:1-17.doi:10.1016/j.steroids.2016.12.014. doi: S0039-128X(17)30002-8 pmid: 28063793
[24]	Bauer M P, Bridgham J T, Langenau D M, Johnson A L, Goetz F W. Conservation of steroidogenic acute regulatory(StAR)protein structure and expression in vertebrates[J]. Molecular and Cellular Endocrinology, 2000, 168(1/2):119-125.doi:10.1016/S0303-7207(00)00316-6. doi: 10.1016/S0303-7207(00)00316-6 URL
[25]	杨沛方, 周志楠, 敖叶, 陈祥. 黔北麻羊StAR基因的克隆、生物信息学分析及在单、多羔黔北麻羊不同性腺组织中的表达[J]. 中国畜牧杂志, 2020, 56(11):77-84.doi:10.19556/j.0258-7033.20200117-03. doi: 10.19556/j.0258-7033.20200117-03
	Yang P F, Zhou Z N, Ao Y, Chen X. Cloning,bioinformatics analysis and expression of StAR gene in Qianbei brown goat[J]. Chinese Journal of Animal Science, 2020, 56(11):77-84.
[26]	Bose M, Whittal R M, Miller W L, Bose H S. Steroidogenic activity of StAR requires contact with mitochondrial VDAC1 and phosphate carrier protein[J]. Journal of Biological Chemistry, 2008, 283(14):8837-8845.doi:10.1074/jbc.M709221200. doi: 10.1074/jbc.M709221200 pmid: 18250166

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