基于转录组测序挖掘槐山羊高繁关键候选基因

doi:10.11843/j.issn.0366-6964.2023.12.018

摘要/Abstract

摘要： 为了探究山羊繁殖性状的内在分子调控机制以及基因调控网络。本研究选取健康无病的3岁左右、体格大小相似的第3胎次母羊,依据繁殖性能记录,采集3只平均胎产羔数为1只的母羊为单羔组,采集3只平均胎产羔数大于2只的母羊为多羔组。采用转录组测序技术进行mRNA文库构建、单羔组与多羔组间差异表达基因筛选及功能鉴定,通过实时荧光定量PCR技术验证基因表达量。本研究构建了6个槐山羊卵巢组织mRNA文库,平均reads总数为41 163 239条,clean reads数为37 908 182条。差异表达分析在多羔与单羔组间共发现121个差异表达基因(P<0.05),其中上调基因30个,下调基因91个。PTX3、MMP13、PAK1、ADAMTS1、COL1A2、CCN1、SLC4A10、FOS、NR4A1、NR4A2和ADCY8等11个基因在单羔与多羔组间表达差异显著(P<0.05),且参与组织器官结构发育功能及内分泌系统醛固酮、皮质醇、催产素和促肾上腺皮质激素等激素分泌;在上述11个基因中共发现236个SNPs,其中7个SNPs为错义突变,4个位于ADAMTS1基因,1个位于PTX3基因,2个位于CCN1基因。本研究结果表明,11个基因在单羔组与多羔组间表达量差异显著,且参与组织器官结构发育及内分泌系统的激素分泌,可能与山羊卵泡发育、排卵及类固醇激素分泌有关,为阐明槐山羊多胎性状遗传机制提供科学依据。

关键词: 槐山羊, 产羔数, 转录组, 差异表达基因

Abstract: The study aimed to explore molecular regulatory mechanism and genes regulatory network of goat reproductive traits. In this study, 3-year-old healthy ewes of 3rd parity with similar body size were selected. According to the reproductive performance records, 3 ewes with 1 lamb on average were collected as single-lamb group, and 3 ewes with more than 2 lambs on average were collected as multi-lamb group. Transcriptome sequencing technology was used to construct mRNA library, screen differentially expressed genes (DEGs) and conduct functional enrichment analysis between single-lamb group and multi-lamb group. Real-time fluorescence quantitative PCR was used to verify gene expression. Six mRNA libraries of ovarian tissues from Huai goats were constructed, with a total number of 41 163 239 raw reads and 37 908 182 clean reads on average. A total of 121 DEGs were found between single-lamb group and multi-lamb group (P<0.05), including 30 up-regulated genes and 91 down-regulated genes. The expressions of PTX3, MMP13, PAK1, ADAMTS1, COL1A2, CCN1, SLC4A10, FOS, NR4A1, NR4A2 and ADCY8 genes were significantly differentially expressed between single-lamb group and multi-lamb group (P<0.05). And these 11 DEGs were involved in the organs development and the secretion of aldosterone, cortisol, oxytocin and adrenocorticotropic hormone in the endocrine system. A total of 236 SNPs were screened in the above 11 DEGs. Seven SNPs were nonsynonymous mutations, among which, 4 SNPs were detected in ADAMTS1 gene, one was in PTX3 gene, and two were in CCN1 gene. The results showed that the expression of 11 genes were significantly different between single-lamb group and multi-lamb group. These DEGs were involved tissues and organs development and hormone secretion in endocrine system. These DEGs may be related to goat follicular development, ovulation and steroid hormone secretion. The results will provide scientific basis for elucidating the genetic mechanism of high fecundity of Huai goat.

Key words: Huai goat, lambing number, transcriptome, differentially expressed genes

中图分类号:

S826.2

韩浩园, 李世凯, 杨瑞巧, 李曼曼, 李君, 哈斯, 赵金艳, 魏红芳, 权凯. 基于转录组测序挖掘槐山羊高繁关键候选基因[J]. 畜牧兽医学报, 2023, 54(12): 5077-5090.

HAN Haoyuan, LI Shikai, YANG Ruiqiao, LI Manman, LI Jun, HA Si, ZHAO Jinyan, WEI Hongfang, QUAN Kai. Mining Key Candidate Genes for High Reproduction Performance of Huai Goats Based on Transcriptome Sequencing[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5077-5090.

参考文献

[1] 王拥庆.槐山羊良种繁育技术推广应用[J].河南畜牧兽医:综合版,2015,36(2):10-12.WANG Y Q.Popularization and application of Huai goat breeding technology[J].Henan Journal of Animal Husbandry and Veterinary Medicine,2015,36(2):10-12.(in Chinese)
[2] 李悦欣,刘爱菊,马晓菲,等.TGFβR1介导TGF-β/Smad信号通路对绵羊颗粒细胞功能的影响[J].畜牧兽医学报,2023,54(8):3335-3347.LI Y X,LIU A J,MA X F,et al.Effect of TGFβR1 on the function of sheep granulosa cells mediated by TGF-β/Smad signaling pathway[J].Acta Veterinaria et Zootechnica Sinica,2023,54(8):3335-3347.(in Chinese)
[3] 蔡元,田见晖,王栋,等.妊娠早期饲粮添加不同水平N-氨甲酰谷氨酸改善母羊产羔性能的研究[J].畜牧兽医学报,2021, 52(1): 126-134.CAI Y,TIAN J H,WANG D,et al.Effects of dietary N-carbamylglutamate supplementation on improving lambing performance of ewes during early pregnancy[J].Acta Veterinaria et Zootechnica Sinica,2021,52(1):126-134.(in Chinese)
[4] 肖帆,张利平,吴建平,等.绵羊mtDNA变异与产羔数关联分析[J].畜牧兽医学报,2021,52(7):1831-1844. XIAO F,ZHANG L P,WU J P,et al.Analysis of correlation between mitochondrial DNA variation and litter size in sheep[J].Acta Veterinaria et Zootechnica Sinica,2021,52(7):1831-1844.(in Chinese)
[5] 马晓菲,刘爱菊,韩红叶,等.BMPR-IB基因在寒泊羊群体中的增羔效应、遗传规律及育种应用分析[J].畜牧兽医学报,2020, 51(4):688-700.MA X F,LIU A J,HAN H Y,et al.Analysis of BMPR-IB gene’s effect on litter size,its heredity rule and breeding application in Hanper sheep[J].Acta Veterinaria et Zootechnica Sinica,2020,51(4):688-700.(in Chinese)
[6] 周世卫,吴庭杰,王倩,等.绵羊FecB突变检测方法的建立及高繁滩羊核心群的构建[J].畜牧兽医学报,2022, 53(9):2920-2929.ZHOU S W,WU T J,WANG Q,et al.Establishment of the efficient method to detect the FecB mutation and the construction of the prolific Tan sheep population[J].Acta Veterinaria et Zootechnica Sinica,2022,53(9):2920-2929.(in Chinese)
[7] 杨初蕾,张译元,唐红,等.FecB基因在绵羊繁殖性能上的研究进展[J].畜牧兽医学报,2023,54(4):1370-1380.YANG C L,ZHANG Y Y,TANG H,et al.Research progress of FecB gene on reproductive performance of sheep[J].Acta Veterinaria et Zootechnica Sinica,2023,54(4):1370-1380.(in Chinese)
[8] 赵雪洋,李丹妮,王钰晨,等.东湖杂交羊产羔性状的GWAS分析及候选基因GRID2的验证[J].畜牧兽医学报, 2023:1-12.(2023-09-08).http://kns.cnki.net/kcms/detail/11.1985.s.20230905.1046.004.html.,54(11):4625-4635.ZHAO X Y,LI D N,WANG Y C,et al.GWAS analysis of lambing traits in East Friesian and Hu crossbred sheep and verification of candidate gene GRID2[J].Acta Veterinaria et Zootechnica Sinica,2023:1-12. (2023-09-08). http://kns.cnki.net/kcms/detail/11.1985.s.20230905.1046.004.html.,54(11):4625-4635.(in Chinese)
[9] 周步峰,石福岳,张建军,等.FSHR基因多态性及其与陇东绒山羊产双羔关联性分析[J].中国畜牧杂志,2021,57(8):67-71.ZHOU B F,SHI F Y,ZHANG J J,et al.Polymorphism of FSHR gene and its correlation with double lambs in Long-Dong Cashmere goats[J].Chinese Journal of Animal Science,2021,57(8):67-71.(in Chinese)
[10] 易鸣,张继,袁润,等.RBP4基因多态性与贵州黑山羊产羔性状关联性分析[J].中国畜牧杂志,2020,56(10):41-46.YI M,ZHANG J,YUAN R,et al.Correlation analysis between polymorphism of RBP4 gene and lambing traits in Guizhou black goat[J].Chinese Journal of Animal Science,2020,56(10):41-46.(in Chinese)
[11] 岳畅.辽宁绒山羊高繁候选基因SNP及其调控非编码RNA的表达分析[D].沈阳:沈阳农业大学,2020.YUE C.Expression analysis of SNP and its regulatory noncoding RNA in Liaoning Cashmere goat[D].Shenyang:Shenyang Agricultural University,2020.(in Chinese)
[12] 陶林,贺小云,江炎庭,等.山羊GJB2和GJB6基因多态性与产羔性能的关联分析[J].中国畜牧杂志,2021,57(10):79-83.TAO L,HE X Y,JIANG Y T,et al.Association analvsis on polymorphism of GJB2 and GJB6 genes and kidding performance in goats[J].Chinese Journal of Animal Science,2021,57(10):79-83.(in Chinese)
[13] 王真.陕北白绒山羊MMAF相关基因遗传变异的挖掘及其与产羔数关联研究[D].杨凌:西北农林科技大学,2020.WANG Z.Genetic variation identification of MMAF-related genes and their associations with litter size in Shaanbei White Cashmere goat[D].Yangling:Northwest A&F University,2020.(in Chinese)
[14] 洪磊,陈祥,唐文,等.黔北麻羊SRD5A2基因干扰载体构建及其对产羔相关基因表达的影响[J].畜牧兽医学报,2020,51(12):2980-2990.HONG L,CHEN X,TANG W,et al.Construction of SRD5A2 gene interference vector and its effect on the expression of lambing related genes in Qianbei Ma goats[J].Acta Veterinaria et Zootechnica Sinica,2020,51(12):2980-2990.(in Chinese)
[15] SANGWAN R S,TRIPATHI S,SINGH J,et al.De novo sequencing and assembly of Centella asiatica leaf transcriptome for mapping of structural,functional and regulatory genes with special reference to secondary metabolism[J].Gene,2013,525(1): 58-76.
[16] 李耀坤,许香萍,陆婷婷,等.影响山羊产羔数lncRNA的筛选及功能分析[J].畜牧兽医学报,2020,51(8):1853-1865.LI Y K,XU X P,LU T T,et al.Screening and functional analysis of lncRNA affecting litter size in goats[J].Acta Veterinaria et Zootechnica Sinica,2020,51(8):1853-1865.(in Chinese)
[17] LING Y H,REN C H,GUO X F,et al.Identification and characterization of microRNAs in the ovaries of multiple and uniparous goats (Capra hircus) during follicular phase[J].BMC Genomics,2014,15(1):339.
[18] 王俊杰.济宁青山羊产羔数性状的基因组和卵泡发育的转录组研究[D].杨凌:西北农林科技大学,2020.WANG J J.Genome analysis of litter size trait and transcriptome analysis of follicular development of Jining grey goat[D].Yangling:Northwest A&F University,2020.(in Chinese)
[19] XU L,LIU C L,NA R,et al.Genetic basis of follicle development in Dazu Black goat by whole-transcriptome sequencing[J]. Animals,2021,11(12):3536.
[20] LIU Y,WU X Q,XIE J,et al.Identification of transcriptome differences in goat ovaries at the follicular phase and the luteal phase using an RNA-Seq method[J].Theriogenology,2020,158:239-249.
[21] 张天浩.不同产羔数燕山绒山羊卵巢组织差异表达基因的筛选与分析[D].秦皇岛:河北科技师范学院,2022.ZHANG T H.Screening and analysis of differentially expressed genes in ovary tissue of Yanshan Cashmere goat with different lambing numbers[D].Qinhuangdao:Hebei Normal University of Science & Technology,2022.(in Chinese)
[22] 喇永富.利用转录组和蛋白质组测序筛选绵羊多羔基因的研究[D].兰州:甘肃农业大学,2020.LA Y F.Study on screening polytocous genes in sheep based on transcriptome and proteomics sequencing[D].Lanzhou:Gansu Agricultural University,2020.(in Chinese)
[23] 王沛婕.PTX3调控奶山羊卵巢颗粒细胞功能及分子机理的研究[D].杨凌:西北农林科技大学,2021.WANG P J.PTX3 regulates function and molecular mechanism of ovarian granulose cells in dairy goat[D].Yangling:Northwest A&F University,2021.(in Chinese)
[24] WOLAK D,HRABIA A.Tamoxifen-induced alterations in the expression of selected matrix metalloproteinases (MMP-2,-9,-10,and-13) and their tissue inhibitors (TIMP-2 and-3) in the chicken ovary[J].Theriogenology,2020,148:208-215.
[25] WOLAK D,SECHMAN A,HRABIA A.Effect of eCG treatment on gene expression of selected matrix metalloproteinases (MMP-2,MMP-7,MMP-9,MMP-10,and MMP-13) and the tissue inhibitors of metalloproteinases (TIMP-2 and TIMP-3) in the chicken ovary[J].Anim Reprod Sci,2021,224:106666.
[26] HRABIA A,WOLAK D,SECHMAN A.Response of the matrix metalloproteinase system of the chicken ovary to prolactin treatment[J].Theriogenology,2021,169:21-28.
[27] NG Y H,ZHU H,PALLEN C J,et al.Differential effects of interleukin-1β and transforming growth factor-β1 on the expression of the inflammation-associated protein,ADAMTS-1,in human decidual stromal cells in vitro[J].Hum Reprod,2006,21(8): 1990-1999.
[28] 何小龙,李蓓,刘永斌,等.蒙古羊ADAMTS1基因外显子3多态性检测及在卵巢和子宫组织中差异表达分析[J].畜牧兽医学报,2013,44(3):399-406.HE X L,LI B,LIU Y B,et al.The polymorphism detection of exon 3 and differential expression analysis in ovaries and uterus of Mongolian sheep ADAMTS1 gene[J].Acta Veterinaria et Zootechnica Sinica,2013,44(3):399-406.(in Chinese)
[29] SHEN-GUNTHER J,XIA Q Q,STACEY W,et al.Molecular pap smear:validation of HPV genotype and host methylation profiles of ADCY8,CDH8,and ZNF582 as a predictor of cervical cytopathology[J].Front Microbiol,2020,11:595902.
[30] LI M,XUE K,LING J,et al.The orphan nuclear receptor NR4A1 regulates transcription of key steroidogenic enzymes in ovarian theca cells[J].Mol Cell Endocrinol,2010,319(1-2):39-46.
[31] 崔胜,王永,朱江江,等.过表达NR4A1促进山羊皮下脂肪细胞分化[J].畜牧兽医学报,2021,52(5):1258-1266.CUI S,WANG Y,ZHU J J,et al.Overexpression of NR4A1 promotes goat subcutaneous adipocytes differentiation[J].Acta Veterinaria et Zootechnica Sinica,2021,52(5):1258-1266.(in Chinese)
[32] 金心,胡珍华,孙新宇,等.转录因子NR4A2在卵巢上皮性肿瘤组织中的表达及其临床意义[J].现代肿瘤医学,2013,21(12): 2775-2778.JIN X,HU Z H,SUN X Y,et al.Expression of nuclear transcription factor NR4A2 protein in ovarian tumor and its clinical significance[J].Journal of Modern Oncology,2013,21(12):2775-2778.(in Chinese)
[33] 刘林清,李庆岗,苏世广,等.猪NR4A1基因的多态性与产仔数性状的关联分析[J].养猪,2021(2):65-67.LIU L Q,LI Q G,SU S G,et al.Analysis of relationship between polymorphisms of porcine NR4A1 gene with litter size trait[J].Swine Production,2021(2):65-67.(in Chinese)