犏牛和牦牛ESCO2基因的序列特征及其在睾丸中的表达对比分析

doi:10.11843/j.issn.0366-6964.2021.011.014

摘要/Abstract

摘要： 旨在克隆犏牛和牦牛姐妹染色单体内聚建立蛋白2（establishment of sister chromatid cohesion N-acetyltransferase 2，ESCO2）基因，并分析其在不同发育阶段睾丸中的表达与定位，为进一步解析ESCO2在减数分裂过程中的作用机制提供理论依据。本研究以健康雄性犏牛及牦牛为试验动物，根据年龄分为胎牛组（5~6月龄）、幼年组（1~2岁）和成年组（3~4岁），每组各3头。通过RT-PCR技术克隆犏牛和牦牛ESCO2基因并进行生物信息学分析，采用实时荧光定量PCR （quantitative real-time PCR，qRT-PCR）检测ESCO2基因在犏牛不同组织中的表达谱，比较分析ESCO2在犏牛和牦牛不同时期睾丸中的表达规律，利用免疫组织化学（immunohistochemistry，IHC）技术检测ESCO2蛋白的细胞定位和表达差异。结果显示，犏牛ESCO2基因（GenBank登录号：MW198470） CDS区为1 833 bp，编码610个氨基酸，与牦牛相比，犏牛ESCO2序列第301~319位多19个氨基酸，另有3个氨基酸突变；犏牛ESCO2蛋白序列与黄牛的同源性高于其他哺乳动物；ESCO2可能与SMC3、SMC1A、PDS5A、PDS5B、STAG2等蛋白相互作用，互作蛋白功能与姐妹染色单体凝聚、减数分裂细胞周期、DNA修复、细胞分裂和染色体重构等生物学过程相关。ESCO2在犏牛各组织中均有表达，但在睾丸中的相对表达水平显著高于其它组织（P<0.05）；在犏牛睾丸中的表达随年龄增长呈上升趋势，幼年和成年时期犏牛睾丸中ESCO2的表达显著低于同时期牦牛（P<0.05）；IHC染色结果发现，雄性犏牛减数分裂阻滞于初级精母细胞，ESCO2蛋白在犏牛初级精母细胞中无表达并与牦牛存在差异。本研究结果表明，犏牛与牦牛的ESCO2基因、蛋白序列差异较大，且在睾丸发育过程中的表达模式差异显著，这可能是引起雄性犏牛减数分裂阻滞及不育的原因之一，其具体作用机制有待进一步研究。

关键词: 犏牛, ESCO2, 睾丸, 减数分裂, 基因表达

Abstract: The aim of this study was to clone the establishment of sister chromatid cohesion N-acetyltransferase 2(ESCO2) gene, analyze its expression and localization in the testis at different developmental stages in cattle-yak and yak, and provide a theoretical basis for further study the role mechanism of ESCO2 in the male meiosis process. Healthy male cattle-yaks and yaks were used as experimental animals in this study, they were divided into fetal cattle group (5-6 months old), juvenile group (1-2 years old) and adult group (3-4 years old), with 3 animals in each group. The CDS region of ESCO2 gene of cattle-yak and yak were cloned by RT-PCR and its sequence characteristics were analyzed by bioinformatics softwares. qRT-PCR was used to detect the expression patterns of ESCO2 gene in different tissues of cattle-yak. The expression of ESCO2 mRNA in testes at different developmental stages of cattle-yak and yak were detected by qRT-PCR, the differences of cell location and expression of ESCO2 protein were detected by immunohistochemistry (IHC) staining. The results showed that the CDS region of ESCO2 genes in cattle-yak was 1 833 bp(MW198470), it encoded 610 amino acids. Compared with yak, the ESCO2 protein sequence of cattle-yak had more 19 amino acids at position 301-319 aa and there were 3 amino acid mutations. The cattle-yak and cattle had higher homology than other mammals based on the protein sequence of ESCO2. Interaction network showed that ESCO2 mainly interacted with SMC3, SMC1A, PDS5A, PDS5B, STAG2 and these proteins were related to sister chromatid condensation, meiotic cell cycle, DNA repair, cell division and chromosome remodeling. ESCO2 was expressed in all tissues of the cattle-yak, but its relative expression level in the testis was significantly higher than those in other tissues (P<0.05). In addition, the spatial expression of ESCO2 in testes of cattle-yak showed an upward trend with age, and the relative expression abundance of ESCO2 in cattle-yak at juvenile and adult stages was significantly lower than that of yak(P<0.05). IHC staining showed that meiosis of male cattle-yak was blocked in primary spermatocytes and ESCO2 protein was differentially expressed in primary spermatocytes between cattle-yak and yak. The research suggested that there was obvious difference in the sequence and expression pattern of ESCO2 between cattle-yak and yak, which may be one of reasons caused male sterility in cattle-yak, but the specific mechanism need further study.

Key words: cattle-yak, ESCO2, testis, meiosis, gene expression

中图分类号:

S823.85

闵星宇, 杨丽雪, 杨满珍, 胡宇磊, 于海玲, 杨璐瑜, 李键, 熊显荣. 犏牛和牦牛ESCO2基因的序列特征及其在睾丸中的表达对比分析[J]. 畜牧兽医学报, 2021, 52(11): 3126-3136.

MIN Xingyu, YANG Lixue, YANG Manzhen, HU Yulei, YU Hailing, YANG Luyu, LI Jian, XIONG Xianrong. Sequence Characteristics and Comparative Expression Analysis in Testis of ESCO2 Gene in Cattle-yak and Yak[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(11): 3126-3136.

参考文献 40

[1]	WU Y, ZHANG W X, ZUO F, et al. Comparison of mRNA expression from Y-chromosome X-degenerate region genes in taurine cattle, yaks and interspecific hybrid bulls[J].Anim Genet, 2019, 50(6):740-743.
[2]	XU C F, SHAH M A, MIPAM T, et al. Bovid microRNAs involved in the process of spermatogonia differentiation into spermatocytes[J].Int J Biol Sci, 2020, 16(2):239-250.
[3]	王泳杰, 王之盛, 胡瑞, 等. 不同品种肉牛产肉性能、牛肉营养品质及风味物质的比较[J].动物营养学报, 2019, 31(8):3621-3631.WANG Y J, WANG Z S, HU R, et al. Comparison of meat performance, nutritional quality and flavor substance in beef of different breeds cattle[J].Chinese Journal of Animal Nutrition, 2019, 31(8):3621-3631.(in Chinese)
[4]	QIU Q, ZHANG G J, MA T, et al. The yak genome and adaptation to life at high altitude[J].Nat Genet, 2012, 44(8):946-949.
[5]	阿果约达, 熊显荣, 王艳, 等. 犏牛雄性不育相关lncRNA的鉴定与分析[J].畜牧兽医学报, 2019, 50(3):551-561.AGUO Y D, XIONG X R, WANG Y, et al. Identification and analysis of long non-coding RNA associated with cattle-yak male infertility[J].Acta Veterinaria et Zootechnica Sinica, 2019, 50(3):551-561.(in Chinese)
[6]	ALOMER R M, DA SILVA E M L, CHEN J R, et al. Esco1 and Esco2 regulate distinct cohesin functions during cell cycle progression[J].Proc Natl Acad Sci U S A, 2017, 114(37):9906-9911.
[7]	BENDER D, DA SILVA E M L, CHEN J R, et al. Multivalent interaction of ESCO2 with the replication machinery is required for sister chromatid cohesion in vertebrates[J].Proc Natl Acad Sci U S A, 2020, 117(2):1081-1089.
[8]	LADURNER R, KREIDL E, IVANOV M P, et al. Sororin actively maintains sister chromatid cohesion[J].EMBO J, 2016, 35(6):635-653.
[9]	MFAREJ M G, SKIBBENS R V.An ever-changing landscape in Roberts syndrome biology:implications for macromolecular damage[J].PLoS Genet, 2020, 16(12):e1009219.
[10]	MCKAY M J, CRAIG J, KALITSIS P, et al. A Roberts syndrome individual with differential genotoxin sensitivity and a DNA damage response defect[J].Int J Radiat Oncol Biol Phys, 2019, 103(5):1194-1202.
[11]	GORDILLO M, VEGA H, TRAINER A H, et al. The molecular mechanism underlying Roberts syndrome involves loss of ESCO2 acetyltransferase activity[J].Hum Mol Genet, 2008, 17(14):2172-2180.
[12]	KOUZNETSOVA E, KANNO T, KARLBERG T, et al. Sister chromatid cohesion establishment factor ESCO1 operates by substrate-assisted catalysis[J].Structure, 2016, 24(5):789-796.
[13]	HOGARTH C A, MITCHELL D, EVANOFF R, et al. Identification and expression of potential regulators of the mammalian mitotic-to-meiotic transition[J].Biol Reprod, 2011, 84(1):34-42.
[14]	LI H, YOU L J, TIAN Y F, et al. DPAGT1-mediated protein N-Glycosylation is indispensable for oocyte and follicle development in mice[J].Adv Sci (Weinh), 2020, 7(14):2000531.
[15]	LALDINSANGI C, VIJAYAPRASADARAO K, RAJAKUMAR A, et al. Two-dimensional proteomic analysis of gonads of air-breathing catfish, Clarias batrachus after the exposure of endosulfan and malathion[J].Environ Toxicol Pharmacol, 2014, 37(3):1006-1014.
[16]	EVANS E B, HOGARTH C, EVANOFF R M, et al. Localization and regulation of murine Esco2 during male and female meiosis[J].Biol Reprod, 2012, 87(3):61.
[17]	LU Y J, CHEN Y, CUI Z K, et al. Distinct roles of cohesin acetyltransferases Esco1 and Esco2 in porcine oocyte meiosis I[J].Cell Cycle, 2019, 18(19):2481-2494.
[18]	LU Y J, DAI X X, ZHANG M Q, et al. Cohesin acetyltransferase Esco2 regulates SAC and kinetochore functions via maintaining H4K16 acetylation during mouse oocyte meiosis[J].Nucleic Acids Res, 2017, 45(16):9388-9397.
[19]	MCNICOLL F, KÜHNEL A, BISWAS U, et al. Meiotic sex chromosome cohesion and autosomal synapsis are supported by Esco2[J].Life Sci Alliance, 2020, 3(3):e201900564.
[20]	LUENSE L J, DONAHUE G, LIN-SHIAO E, et al. Gcn5-mediated histone acetylation governs nucleosome dynamics in spermiogenesis[J].Dev Cell, 2019, 51(6):745-758.e6.
[21]	AKSNES H, REE R, ARNESEN T.Co-translational, post-translational, and non-catalytic roles of N-terminal acetyltransferases[J]. Mol Cell, 2019, 73(6):1097-1114.
[22]	CHEN Y, ZHOU Y L, YIN H S.Recent advances in biosensor for histone acetyltransferase detection[J].Biosens Bioelectron, 2021, 175:112880.
[23]	RIVERA-COLÓN Y, MAGUIRE A, LISZCZAK G P, et al. Molecular basis for cohesin acetylation by establishment of sister chromatid cohesion N-acetyltransferase ESCO1[J].J Biol Chem, 2016, 291(51):26468-26477.
[24]	ALEXEEV V, IGOUCHEVA O, DOMASHENKO A, et al. Localized in vivo genotypic and phenotypic correction of the albino mutation in skin by RNA-DNA oligonucleotide[J].Nat Biotechnol, 2000, 18(1):43-47.
[25]	KOBLAN L W, ERDOS M R, WILSON C, et al. In vivo base editing rescues Hutchinson-Gilford progeria syndrome in mice[J].Nature, 2021, 589(7843):608-614.
[26]	PECKER L H, LANZKRON S.Sickle cell disease[J].Ann Intern Med, 2021, 174(1):ITC1-ITC16.
[27]	VEGA H, WAISFISZ Q, GORDILLO M, et al. Roberts syndrome is caused by mutations in ESCO2, a human homolog of yeast ECO1 that is essential for the establishment of sister chromatid cohesion[J].Nat Genet, 2005, 37(5):468-470.
[28]	CHENG H Z, ZHANG N G, PATI D.Cohesin subunit RAD21:from biology to disease[J].Gene, 2020, 758:144966.
[29]	ZHANG J, FENG C, SU H D, et al. The cohesin complex subunit ZmSMC3 participates in meiotic centromere pairing in maize[J].Plant Cell, 2020, 32(4):1323-1336.
[30]	AL-JOMAH N, MUKOLOLO L, ANJUM A, et al. Pds5A and Pds5B display non-redundant functions in mitosis and their loss triggers Chk1 activation[J].Front Cell Dev Biol, 2020, 8:531.
[31]	DEARDORFF M A, BANDO M, NAKATO R, et al. HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle[J].Nature, 2012, 489(7415):313-317.
[32]	KULESZEWICZ K, FU X W, KUDO N R.Cohesin loading factor Nipbl localizes to chromosome axes during mammalian meiotic prophase[J].Cell Div, 2013, 8(1):12.
[33]	FAGERBERG L, HALLSTRÖM B M, OKSVOLD P, et al. Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics[J].Mol Cell Proteomics, 2014, 13(2):397-406.
[34]	YU Y, FUSCOE J C, ZHAO C, et al. A rat RNA-Seq transcriptomic BodyMap across 11 organs and 4 developmental stages[J]. Nat Commun, 2014, 5:3230.
[35]	CHEN H M, ZHANG L, HE W T, et al. ESCO2 knockdown inhibits cell proliferation and induces apoptosis in human gastric cancer cells[J].Biochem Biophys Res Commun, 2018, 496(2):475-481.
[36]	GUO X B, HUANG B, PAN Y H, et al. ESCO2 inhibits tumor metastasis via transcriptionally repressing MMP2 in colorectal cancer[J].Cancer Manag Res, 2018, 10:6157-6166.
[37]	LOU Y N, LIU W J, WANG C L, et al. Histological evaluation and Prdm9 expression level in the testis of sterile male cattle-yaks[J].Livest Sci, 2014, 160:208-213.
[38]	SATO Y, KURIWAKI R, HAGINO S, et al. Abnormal functions of Leydig cells in crossbred cattle-yak showing infertility[J]. Reprod Domest Anim, 2020, 55(2):209-216.
[39]	YAN P, XIANG L, GUO X, et al. The low expression of Dmrt7 is associated with spermatogenic arrest in cattle-yak[J].Mol Biol Rep, 2014, 41(11):7255-7263.
[40]	李波, 庄梦茹, 李娜, 等. 奶山羊ESCO2基因克隆及其功能的初步研究[J].农业生物技术学报, 2016, 24(2):176-185.LI B, ZHUANG M R, LI N, et al. Molecular cloning of ESCO2 gene and the preliminary study of its function in dairy goat (Capra hircus)[J].Journal of Agricultural Biotechnology, 2016, 24(2):176-185.(in Chinese)