藏羊MSTN基因克隆、生物信息学及组织表达分析

doi:10.16431/j.cnki.1671-7236.2022.11.021

摘要/Abstract

摘要： 【目的】对藏羊肌肉生长抑制素（myostatin，MSTN）基因进行克隆和生物信息学分析，检测其在藏羊不同组织中的表达，为探究MSTN基因在藏羊中的生物学功能提供参考。【方法】以藏羊背最长肌组织cDNA为模板，克隆藏羊MSTN基因完整CDS区序列并测序，用SeqMan程序对测序结果进行拼接，并用BLAST在线程序对组装后的序列进行分析鉴定。用生物信息学软件进行相似性比对、系统进化树构建及生物信息学分析，用实时荧光定量PCR检测MSTN基因在藏羊不同组织中的表达量。【结果】藏羊MSTN基因CDS全长为1 128 bp，编码375个氨基酸。藏羊MSTN基因氨基酸序列与绵羊、牦牛、牛、猪、恒河猴、人、黑猩猩、犬、鸡及斑马的相似性依次为100.0%、93.4%、93.4%、95.2%、94.4%、94.2%、94.4%、93.1%、87.8%和87.5%；系统进化树分析结果表明，藏羊与绵羊的亲缘关系最近，与斑马和鸡的亲缘关系最远。藏羊MSTN蛋白属于亲水性分泌蛋白，且具有不稳定性，不含跨膜结构，含1个信号肽，存在31个潜在的磷酸化位点、2个N-糖基化修饰位点，主要分布在线粒体和细胞质中；MSTN蛋白二级结构以无规卷曲为主，其次是α-螺旋、延伸链和β-转角；三级结构预测结果与二级结构一致。实时荧光定量PCR结果显示，MSTN基因在藏羊不同组织中均有表达，其中在臂三头肌和半腱肌的表达量显著高于肺脏、下丘脑、心脏、肝脏、十二指肠、瘤胃和肾脏（P<0.05）。【结论】成功克隆了藏羊MSTN基因；该基因在藏羊肌肉中的表达量高于内脏组织。该结果为进一步研究MSTN基因对藏羊肌肉生长发育的调控机制奠定了基础。

关键词: MSTN基因; 克隆; 生物信息学分析; 基因表达

Abstract: 【Objective】 The objective of this study was to clone and bioinformatic analysis of myostatin (MSTN) gene in Tibetan sheep, and investigate its expression in different tissues of Tibetan sheep, so as to provide a referencce for exploring the biological function of MSTN gene in Tibetan sheep.【Method】 Using cDNA of longissimus dorsi muscle of Tibetan sheep as template, the complete CDS region of MSTN gene of Tibetan sheep was cloned and sequenced, and the sequencing results were spliced by SeqMan program, and the assembled sequence was analyzed and identified by BLAST online procedure.Similarity comparison, phylogenetic tree construction and bioformztics analysis were carried out by bioinformatics softwares, and the expression of MSTN gene in different tissues of Tibetan sheep was detected by Real-time quantitative PCR.【Result】 The CDS length of MSTN gene in Tibetan sheep was 1 128 bp and encode 375 amino acids.The amino acid sequence similarity of MSTN gene in Tibetan sheep were 100.0%, 93.4%, 93.4%, 95.2%, 94.4%, 94.2%, 94.4%, 93.1%, 87.8% and 87.5% with Ovis aries, Bos mutus, Bos taurus, Sus scrofa, Macaca mulatta, Homo sapiens, Pan troglodytes, Canis lupus familiaris, Gallus gallus and Maylandia zebra.The results of phylogenetic tree analysis showed that Tibetan sheep was the closest to Ovis aries and the furthest to Maylandia zebra and Gallus gallus. MSTN protein of Tibetan sheep was a hydrophilic secretory protein, and had instability, without transmembrane structure, contains 1 signal peptide, has 31 potential phosphorylation sites and 2 N-glycosylation modification sites, it was mainly distributed in mitochondria and cytoplasm.The secondary structure of MSTN protein was consistent with the prediction of tertiary structure, mainly random coil, followed by α-helix, extended chain and β-turn, which was a mixed protein.Real-time quantitative PCR results showed that MSTN gene was expressed in hypothalamus, pituitary, heart, liver, spleen, lungs, kidneys, rumen, duodenum, triceps brachio, semitendinosus, quadriceps femoris and longissimus dorsi, and the MSTN gene was expressed in different tissues of Tibetan sheep, and the expression of MSTN gene in triceps brachii and semitendinosus muscle was significantly higher than that in lung, hypothalamus, heart, liver, duodenum, rumen and kidney(P<0.05).【Conclusion】 MSTN gene was successfully cloned from Tibetan sheep, and its expression in muscle was higher than that in visceral tissue, which laid a foundation for further study of the regulatory mechanism of MSTN gene on muscle growth and development in Tibetan sheep.

Key words: MSTN gene; cloning; bioinformatics analysis; gene expression

中图分类号:

S826.8

张军霞, 贺娜, 李明明, 吕彩玲, 崔亚杰, 郎侠, 王彩莲. 藏羊MSTN基因克隆、生物信息学及组织表达分析[J]. 中国畜牧兽医, 2022, 49(11): 4307-4317.

ZHANG Junxia, HE Na, LI Mingming, LYU Cailing, CUI Yajie, LANG Xia, WANG Cailian. Cloning,Bioinformatics and Tissue Expression Analysis of MSTN Gene in Tibetan Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4307-4317.

参考文献

[1] MCPHERRON A C, LAWLER A M, LEE S J.Regulation of skeletal muscle mass in mice by a new TGF-superfamily member[J].Nature, 1997, 387(6628):83-90.
[2] THOMAS M, LANGLEY B, BERRY C, et al.Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation[J].The Journal of Biological Chemistry, 2000, 275(51):40235-40243.
[3] LANGLEY B, THOMAS M, BISHOP A, et a1.Myostatin inhibits myoblast differentiation by down-regulating MyoD expression[J].The Journal of Biological Chemistry, 2002, 277(51):49831-49840.
[4] DENG B, ZANG F, WEN J, et al.The function of myostatin in the regulation of fat mass in mammals[J].Nutrition & Metabolism, 2017, 14(1):29.
[5] WANG X, NIU Y, ZHOU J, et al.CRISPR/Cas9-mediated MSTN disruption and heritable mutagenesis in goats causes increased body mass[J].Animal Genetics, 2018, 49(1):43-51.
[6] MCPHERRON A C, LEE S J.Suppression of body fat accumulation in myostatin-deficient mice[J]. The Journal of Clinical Investigation, 2002, 109(5):595-601.
[7] LEE S J, MCPHERRON A C.Regulation of myostatin activity and muscle growth[J]. Proceedings of the National Academy of Sciences of the United States of America, 2001, 98(16):9306-9311.
[8] WHITTEMORE L A, SONG K, LI X, et al.Inhibition of myostatin in adult mice increases skeletal muscle mass and strength[J].Biochemical and Biophysical Research Communications, 2003, 300(4):965-971.
[9] 毕瀚方.MSTN基因编辑梅山猪背最长肌lncRNA的鉴定与功能分析[D].北京:中国农业科学院, 2021. BI H F.Identification and functional of lncRNA in the longisimus dorsi muscle of MSTN edited Meishan pigs[D].Beijing:Chinese Academy of Agricultural Sciences, 2021.(in Chinese)
[10] MICHEL G, JEANPLONG F, SHARMA M, et a1.Genomic organization and neonatal expression of the bovine myostatin gene[J].Molecular and Cellular Biochemistry, 2001, 200:31-37.
[11] JAKARIA J, WENN L N A, RIYADI I, et al.Discovery of SNPs and indel 11-bp of the myostatin gene and its association with the double-muscled phenotype in Belgian Blue crossbred cattle[J].Gene, 2021, 784:145598.
[12] ZHOU Z W, CAO G H, LI Z, et al.Truncated gRNA reduces CRISPR/Cas9-mediated off-target rate for MSTN gene knockout in bovines[J].Journal of Integrative Agriculture, 2019, 12(18):2835-2843.
[13] 刘皎.MSTN基因敲除绒山羊生产性能及分子生物学评价[D].杨凌:西北农林科技大学, 2020. LIU J.Production performance and molecular biology evaluation of MSTN gene knockout cashmere goat[D].Yangling:Northwest A&F University, 2020.(in Chinese)
[14] TRUKHACHEV V, YATSYK O, TELEGINA E, et al.Comparison of the myostatin (MSTN) gene in Russian Stavropol Merino sheep and New Zealand Merino sheep[J].Small Ruminant Research, 2018, 160:103-106.
[15] GROCHOWSKA E, BORYS B, LISIAK D, et al.Genotypic and allelic effects of the myostatin gene (MSTN) on carcass, meat quality, and biometric traits in Colored Polish Merino sheep[J].Meat Science, 2019, 151:4-17.
[16] LI W J, LI R Y, WEI Y H, et al.Effect of MSTN mutation on growth and carcass performance in Duroc×Meishan hybrid population[J]. Animals (Basel), 2020, 6:932.
[17] XIN X B, YANG S P, LI X, et al.Proteomics insights into the effects of MSTN on muscle glucose and lipid metabolism in genetically edited cattle[J].General and Comparative Endocrinology, 2020, 291:1132-1137.
[18] ARIKAWA E, SUN Y, WANG J, et al.Cross-platform comparison of SYBR^® Green Real-time PCR with TaqMan PCR, microarrays and other gene expression measurement technologies evaluated in the MicroArray Quality Control (MAQC)study[J]. BMC Genomics, 2008, 9:328.
[19] 朱秋宇, 胡兰.动物机体中肌肉生长抑制素基因的研究进展[J].现代畜牧兽医, 2014, 9:51-55. ZHU Q Y, HU L.Research progress of myostatin gene in animal[J].Modern Animal Husbandry and Veterinary Medicine, 2014, 9:51-55.(in Chinese)
[20] 金鑫燕.山羊肌生成抑制素(MSTN)基因的克隆及生物信息学分析[J].中国畜牧兽医, 2011, 38(9):111-114. JIN X Y.Cloning and bioinformatics analysis of myostatin (MSTN) gene in goat[J]. China Animal Husbandry & Veterinary Medicine, 2011, 38(9):111-114.(in Chinese)
[21] 任磊, 帅素容, 杨显彬, 等.藏猪肌肉生长抑制素基因外显子1的PCR-SSCP分析[J].湖北农业科学, 2007, 4:517-519. REN L, SHUAI S R, YANG X B, et al.Analysis of exon 1 of myostatin gene in Tibetan pigs by PCR-SSCP[J].Hubei Agricultural Sciences, 2007, 4:517-519.(in Chinese)
[22] 张丽, 刘丽霞, 李强子, 等.天祝白牦牛MSTN基因编码区克隆及生物信息学分析[J].浙江农业学报, 2017, 29(4):618-624. ZHANG L, LIU L X, LI Q Z, et al.Cloning and bioinformatics analysis of MSTN gene encoding region in Tianzhu White yak[J].Acta Agriculturae Zhejiangensis, 2017, 29(4):618-624.(in Chinese)
[23] 姜懿轩, 王亚玲, 邢珊珊, 等.新西兰白兔MSTN基因克隆、生物信息学分析及表达谱构建[J].中国畜牧兽医, 2021, 48(10):3501-3511. JIANG Y X, WANG Y L, XING S S, et al.Cloning, bioinformatics analysis and expression profiling of MSTN gene in New Zealand White rabbit[J].China Animal Husbandry & Veterinary Medicine, 2021, 48(10):3501-3511.(in Chinese)
[24] 王梦雅, 赵学亮, 徐明, 等.山羊紧密连接相关蛋白ZO-1序列的生物信息学分析[J].黑龙江畜牧兽医, 2018, 21:1-4. WANG M Y, ZHAO X L, XU M, et al.Bioinformatics analysis of ZO-1 sequence of tight junction correlation protein in goat[J].Heilongjiang Animal Husbandry and Veterinary Medicine, 2018, 21:1-4.(in Chinese)
[25] 阮班军, 代鹏, 王伟, 等.蛋白质翻译后修饰研究进展[J].中国细胞生物学学报, 2014, 36(7):1027-1037. RUAN B J, DAI P, WANG W, et al.Research progress on post-translational modification of proteins[J].Chinese Journal of Cell Biology, 2014, 36(7):1027-1037.(in Chinese)
[26] 王真, 潘赟, 毕谊, 等.动物MSTN基因作用机制及突变研究[J].中国牛业科学, 2019, 45(4):22-32. WANG Z, PAN Y, BI Y, et al.Research on the mechanism and mutation of MSTN gene in animal[J].China Cattle Science, 2019, 45(4):22-32.(in Chinese)
[27] ROBERT S B, GOETZ F W.Differential skeletal muscle expression of myostatin across teleost species and the isolation of multiple myostatin isoforms[J].FEBS Letters, 2001, 491(3):212-216.
[28] CIAMELA P, WIATER E, SITH S M, et al.Presence, actions, and regulation of myostatin in rat uterus and myometrial cells[J]. Endocrinology, 2009, 150(2):906-914.
[29] DEGAI G.Expression of the growth hormone and insulin-like growth factor 1 genes in male and female Blue Gourami (Trichogaster trichopterus) at different temperatures[J].Journal of Asian Scientific Research, 2014, 4(8):413-427.
[30] SATYENDRA P S, PRIYA K, ROHIT K, et al.Molecular characterization and phylogeny based analysis of complete coding sequence of myostatin (MSTN) gene in Indian goat breeds[J].Small Ruminant Research, 2014, 2(116):100-110.
[31] 马晓燕, 祖玲玲, 吐来力江·哈木太, 等.新疆也木勒白羊MSTN基因在不同组织中的表达定量研究[J].中国草食动物科学, 2015, 35(5):4-7. MA X Y, ZU L L, TULAILIJIANG H M T, et al.Quantitative expression of MSTN gene in different tissues of Xinjiang Yammu White sheep[J]. China Herbivore Science, 2015, 35(5):4-7.(in Chinese)
[32] 杨家大, 彭舒.贵州地方山羊品种肌肉生长抑制素基因的表达差异[J].江苏农业学报, 2015, 31(2):369-375. YANG J D, PENG S.Expression of myostatin gene in Guizhou goat breeds[J].Journal of Jiangsu Agricultural Sciences, 2015, 31(2):369-375.(in Chinese)
[33] 梁婧娴, 陈志成, 郑玉才, 等.藏系绵羊MSTN基因在不同年龄不同组织的表达定量研究[J].安徽农业科学, 2011, 39(17):10454-10457. LIANG J X, CHEN Z C, ZHENG Y C, et al.Quantitative study on expression of MSTN gene in different tissues of Tibetan sheep at different ages[J].Journal of Anhui Agricultural Sciences, 2011, 39(17):10454-10457.(in Chinese)