绵羊MYL基因家族的鉴定与组织表达分析

doi:10.11843/j.issn.0366-6964.2024.04.019

摘要/Abstract

摘要： 为了解肌球蛋白轻链(myosin light chain，MYL)基因家族在绵羊中的分子进化关系及表达变化，本研究利用生物信息学方法对绵羊MYL基因家族进行鉴定和系统分析。各选取了3只军垦白肉羊、新疆细毛羊和湖羊成年公羊各组织样品，采用qRT-PCR方法验证家族成员MYL1、MYL6B和MYLPF基因的表达情况。结果显示，12个MYL基因分布在9条染色体上，编码由147~211氨基酸组成的亲水性蛋白质，无信号肽和跨膜结构域，二级结构以α-螺旋为主。根据基因结构、保守结构域和基序分布等特征，可以将这些基因家族成员分为3个组。共线性分析结果表明，MYL2和MYL10、MYL2和MYLPF存在片段复制，且这些片段受到较强的选择压力。蛋白互作网络显示MYH10与绵羊MYL家族成员均存在互作关系。MYLs的组织表达模式差异较大，其中MYL1、MYL2、MYL6B和MYLPF在骨骼肌组织中高水平表达。基因克隆测序结果显示，扩增得到目的基因的CDS全长，qRT-PCR结果表明，MYL1、MYL6B和MYLPF在军垦白肉羊肌肉组织中的表达水平显著高于其他组织(P < 0.000 1)，并且在军垦白肉羊骨骼肌中的表达水平显著高于湖羊和细毛羊(P < 0.000 1)。以上结果为深入研究绵羊MYL基因家族的功能以及优化绵羊肉用性能提供了线索。

关键词: 绵羊, MYL, 基因家族, 生物信息学分析, 基因表达

Abstract: The purpose of this study was to understand the molecular evolutionary relationships and expression level of the MYL gene family in sheep. In this study, the bioinformatics methods were utilized to identify and analyze the MYL gene family in sheep. The tissue samples were collected from 3 adult males of Junken white meat sheep, Xinjiang fine-wool sheep and Hu sheep, respectively. Quantitative real-time PCR (qRT-PCR) was used to detect the expression level of MYL1, MYL6B, and MYLPF genes in various tissue of Junken white meat sheep, Xinjiang fine-wool sheep and Hu sheep. The results showed that 12 MYL gene family members were located on 9 different chromosomes. They encoded hydrophilic and varying lengths proteins consisting of 147-211 amino acids. These proteins lacked signal peptides and transmembrane domains and predominantly exhibited α-helices as the secondary structure. Phylogenetic analysis revealed that MYLs were divided into 3 groups, and the members located in the same group had similar gene structures, conserved domains and motif distributions. Collinearity analysis indicated that MYL2 had segmental duplications with MYL10 and MYLPF, and experienced strong purifying selection. Protein-protein interaction network analysis revealed interactions between MYH10 and other members. Tissue expression profile analysis showed that the expression patterns of MYL genes had significant differences. The MYL1, MYL2, MYL6B, and MYLPF displayed high expression levels in skeletal muscle tissue. The results of gene cloning and sequencing showed that the full-length CDS of the target gene was amplified. The expression levels of MYL1, MYL6B and MYLPF in muscle tissues of Junken white meat sheep were significantly higher than those in other tissues (P < 0.000 1). And the 3 genes were dominant expressions in skeletal muscles of Junken white meat sheep than those in Hu sheep and Fine-wool sheep (P < 0.000 1). The results of the study provide valuable insights for further investigation into the functionality of the MYL gene family in sheep and the improvement of sheep meat performance traits.

Key words: sheep, MYL, gene family, bioinformatics analysis, gene expression

中图分类号:

S826.2

杨杨, 余乾, 刘昱成, 杨华, 赵卓, 王立民, 周平, 杨庆勇, 代蓉. 绵羊MYL基因家族的鉴定与组织表达分析[J]. 畜牧兽医学报, 2024, 55(4): 1551-1564.

YANG Yang, YU Qian, LIU Yucheng, YANG Hua, ZHAO Zhuo, WANG Limin, ZHOU Ping, YANG Qingyong, DAI Rong. Identification and Tissue Expression Analysis of the Sheep MYL Gene Family[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1551-1564.

参考文献

[1] PRACHE S, SCHREURS N, GUILLIER L. Review:factors affecting sheep carcass and meat quality attributes[J]. Animal, 2022, 16(Suppl):100330.
[2] YANG Y W, ZHOU Y X, SUN Z P. Review on the influencing factors and control measures of mutton quality[J]. Acta Ecologae Animalis Domastici, 2013, 34(12):82-85. (in Chinese)
杨宇为, 周玉香, 孙占鹏. 羊肉品质的影响因素及其调控措施研究概况[J]. 家畜生态学报, 2013, 34(12):82-85.
[3] XU H G, XU G Y, WANG D H, et al. Molecular cloning, sequence identification and expression analysis of novel caprine MYLPF gene[J]. Mol Biol Rep, 2013, 40(3):2565-2572.
[4] TIMSON D J. Fine tuning the myosin motor:the role of the essential light chain in striated muscle myosin[J]. Biochimie, 2003, 85(7):639-645.
[5] ZHANG C L, WANG G Z, JI Z B, et al. Molecular cloning, characterisation and mRNA expression analysis of the sheep myosin light chain 1 gene[J]. Gene, 2015, 569(1):51-59.
[6] ZHANG S Z, XIE H Q, XU Y, et al. An experimental study of the role of myosin light chain in myogenesis in vitro[J]. Chinese Journal of Reparative and Reconstructive Surgery, 2008, 22(6):753-758. (in Chinese)
张素珍, 解慧琪, 徐永, 等. 肌球蛋白轻链在肌肉再生中作用的体外实验研究[J]. 中国修复重建外科杂志, 2008, 22(6):753-758.
[7] HEISSLER S M, SELLERS J R. Myosin light chains:teaching old dogs new tricks[J]. Bioarchitecture, 2014, 4(6):169-188.
[8] SHI J P, WANG X Y, SONG Y L, et al. Excavation of genes related to the mining of growth, development, and meat quality of two crossbred sheep populations based on comparative transcriptomes[J]. Animals, 2021, 11(6):1492.
[9] MOHAMMADABADI M, BORDBAR F, JENSEN J, et al. Key genes regulating skeletal muscle development and growth in farm animals[J]. Animals, 2021, 11(3):835.
[10] JIAO D, JI K X, WANG W Q, et al. Transcriptome profiles of the liver in two cold-exposed sheep breeds revealed different mechanisms and candidate genes for thermogenesis[J]. Genet Res, 2021, 2021:e10.
[11] ZHAN S Y, ZHAI H F, TANG M, et al. Profiling and functional analysis of mRNAs during skeletal muscle differentiation in goats[J]. Animals (Basel), 2022, 12(8):1048.
[12] VERARDO L L, NASCIMENTO C S, SILVA F F, et al. Identification and validation of differentially expressed genes from pig skeletal muscle[J]. J Anim Breed Genet, 2012, 130(5):372-381.
[13] XIE Y C, RILE N, LI X W, et al. Analysis of cashmere goat meat by label-free proteomics shows that MYL3 is a potential molecular marker of meat toughness[J]. Czech J Anim Sci, 2022, 67(4):137-146.
[14] NOCE A, CARDOSO T F, MANUNZA A, et al. Expression patterns and genetic variation of the ovine skeletal muscle transcriptome of sheep from five Spanish meat breeds[J]. Sci Rep, 2018, 8(1):10486.
[15] PAN C L, WANG S Z, YANG C Y, et al. Genome-wide identification and expression profiling analysis of Wnt family genes affecting adipocyte differentiation in cattle[J]. Sci Rep, 2022, 12(1):489.
[16] CHEN C J, WU Y, LI J W, et al. TBtools-Ⅱ:a "one for all, all for one" bioinformatics platform for biological big-data mining[J]. Mol Plant, 2023, 16(11):1733-1742.
[17] CHEN D N, MA X S, DAI J F, et al. Preliminary study on the function of MGF360-13L gene of African swine fever virus multigene family[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(12):4419-4428. (in Chinese)
陈达年, 马旭升, 代军飞, 等. 非洲猪瘟病毒多基因家族MGF360-13L基因功能的初步研究[J]. 畜牧兽医学报, 2022, 53(12):4419-4428.
[18] JACOB MACHADO D, PORTELLA DE LUNA MARQUES F, JIMÉNEZ-FERBANS L, et al. An empirical test of the relationship between the bootstrap and likelihood ratio support in maximum likelihood phylogenetic analysis[J]. Cladistics, 2022, 38(3):392-401.
[19] NASER-KHDOUR S, QUANG MINH B, LANFEAR R. Assessing confidence in root placement on phylogenies:an empirical study using nonreversible models for mammals[J]. System Biol, 2022, 71(4):959-972.
[20] MA S J, XU Y J, HE K, et al. Molecular evolution and expression patterns of a multigene family of Toll-like receptors in ruminants[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9):3722-3734. (in Chinese)
马淑娟, 徐祎洁, 何珂, 等. 反刍动物Toll样受体多基因家族的分子进化及表达模式分析[J]. 畜牧兽医学报, 2023, 54(9):3722-3734.
[21] CHAVES L D, OSTROSKI B J, REED K M. Myosin light chain genes in the turkey (Meleagris gallopavo)[J]. Cytogenet Genome Res, 2003, 102(1/4):340-346.
[22] LI X, XIE S S, QIAN L L, et al. Identification of genes related to skeletal muscle growth and development by integrated analysis of transcriptome and proteome in myostatin-edited Meishan pigs[J]. J Proteomics, 2020, 213:103628.
[23] LIU R L, WU L, YUAN W, et al. Function of MYL3 gene in muscle growth of cattle[J]. Acta Agriculturae Boreali-Sinica, 2019, 34(2):221-228. (in Chinese)
刘瑞莉, 吴磊, 袁玮, 等. MYL3基因在肉牛肌肉生长过程中的功能研究[J]. 华北农学报, 2019, 34(2):221-228.
[24] WETERMAN M A J, BARTH P G, VAN SPAENDONCK-ZWARTS K Y, et al. Recessive MYL2 mutations cause infantile type Ⅰ muscle fibre disease and cardiomyopathy[J]. Brain, 2013, 136(1):282-293.
[25] HEIZMANN C W. Calcium-binding proteins of the EF-hand superfamily:from basics to medical applications[M]. New York:Springer, 2019.
[26] ZHUANG Z W, XU L Y, YANG J, et al. Weighted single-step genome-wide association study for growth traits in Chinese simmental beef cattle[J]. Genes (Basel), 2020, 11(2):189.
[27] YE M S, YE F, HE L T, et al. Transcriptomic analysis of chicken myozenin 3 regulation reveals its potential role in cell proliferation[J]. PLoS One, 2017, 12(12):e0189476.
[28] YUE C J, LIANG X J, WANG X Q, et al. Whole genome methylation variation analysis of longissimus dorsi between tan sheep and hu sheep[J]. China Animal Husbandry & Veterinary Medicine, 2020, 47(10):3058-3068. (in Chinese)
岳彩娟, 梁小军, 王秀琴, 等. 滩羊和湖羊背最长肌全基因组甲基化差异分析[J]. 中国畜牧兽医, 2020, 47(10):3058-3068.
[29] ADAMEK M, ALANJARY M, ZIEMERT N. Applied evolution:phylogeny-based approaches in natural products research[J]. Nat Prod Rep, 2019, 36(9):1295-1312.
[30] ZHANG C L. Cloning, analysis and protein expression of MYL1 gene promoter region in small tail Han sheep[J]. Chinese Journal of Animal Science, 2020, 56(2):150-152. (in Chinese)
张春兰. 小尾寒羊MYL1基因启动子区的克隆、分析及蛋白表达[J]. 中国畜牧杂志, 2020, 56(2):150-152.
[31] ZHANG M F, LU J W, ZHA L, et al. Cloning and tissue expression analysis of MYL3 gene in yak (Bos grunniens)[J]. Journal of Northwest A & F University (Natural Science Edition), 2024, 52(4):1-9. (in Chinese)
张梦帆, 路建卫, 扎老, 等. 牦牛MYL3基因的克隆及组织表达分析[J]. 西北农林科技大学学报(自然科学版), 2024, 52(4):1-9.
[32] PENG W H, LI M X, LI H L, et al. Dysfunction of myosin light-chain 4(MYL4) leads to heritable atrial cardiomyopathy with electrical, contractile, and structural components:evidence from genetically-engineered rats[J]. J Am Heart Assoc, 2017, 6(11):e007030.
[33] GHAZIZADEH Z, KIVINIEMI T, OLAFSSON S, et al. Metastable atrial state underlies the primary genetic substrate for MYL4 mutation-associated atrial fibrillation[J]. Circulation, 2020, 141(4):301-312.
[34] DEEG C A, DEGROOTE R L, GIESE I M, et al. CD11d is a novel antigen on chicken leukocytes[J]. J Proteomics, 2020, 225:103876.
[35] PARK I, HAN C, JIN S, et al. Myosin regulatory light chains are required to maintain the stability of myosin Ⅱ and cellular integrity[J]. Biochem J, 2011, 434(1):171-180.
[36] NOGUCHI S, TSUKAHARA T, FUJITA M, et al. cDNA microarray analysis of individual duchenne muscular dystrophy patients[J]. Hum Mol Genet, 2003, 12(6):595-600.
[37] CUI H X, LIU R R, ZHAO G P, et al. Identification of differentially expressed genes and pathways for intramuscular fat deposition in pectoralis major tissues of fast-and slow-growing chickens[J]. BMC Genomics, 2012, 13:213.
[38] LIANG S S, PANG C Y, DENG T X, et al. STAT gene family identification in water buffalo[J]. China Cattle Science, 2022, 48(1):45-52. (in Chinese)
梁莎莎, 庞春英, 邓廷贤, 等. 水牛STAT基因家族分析[J]. 中国牛业科学, 2022, 48(1):45-52.
[39] ZHAO S R, YE Z, STANTON R. Misuse of RPKM or TPM normalization when comparing across samples and sequencing protocols[J]. RNA, 2020, 26(8):903-909.
[40] ZHANG C L, WANG J M, WANG G Z, et al. Molecular cloning and mRNA expression analysis of sheep MYL3 and MYL4 genes[J]. Gene, 2016, 577(2):209-214.