中国农业科学 ›› 2021, Vol. 54 ›› Issue (14): 3113-3123.doi: 10.3864/j.issn.0578-1752.2021.14.016
王继卿(),郝志云,沈继源,柯娜,黄兆春,梁维炜,罗玉柱,胡江,刘秀,李少斌
收稿日期:
2020-11-13
接受日期:
2020-02-04
出版日期:
2021-07-16
发布日期:
2021-07-26
作者简介:
王继卿,E-mail: 基金资助:
WANG JiQing(),HAO ZhiYun,SHEN JiYuan,KE Na,HUANG ZhaoChun,LIANG WeiWei,LUO YuZhu,HU Jiang,LIU Xiu,LI ShaoBin
Received:
2020-11-13
Accepted:
2020-02-04
Online:
2021-07-16
Published:
2021-07-26
摘要:
【目的】长链非编码RNA(long non-coding RNAs,lncRNAs)是一类长度大于200 nt的非编码RNA分子,它对奶牛和奶山羊的乳腺发育和泌乳过程发挥了重要调控作用,然而在绵羊上研究甚少。为此开展lncRNAs对绵羊泌乳性能的调控作用研究,为解析绵羊泌乳性能的分子机理提供理论基础。【方法】选取高泌乳性能(高泌乳量、高乳脂率)和低泌乳性能(低泌乳量、低乳脂率)小尾寒羊各3只,采集泌乳期乳腺组织,用RNA-Seq构建lncRNAs表达谱,研究差异表达lncRNAs靶基因的GO和KEGG富集通路,最后用实时荧光定量PCR(reverse transcription-quantitative PCR, RT-qPCR)对16个差异表达lncRNAs进行验证。【结果】在小尾寒羊乳腺组织中共鉴定出7 239个表达的lncRNAs,包括2 262个已知lncRNAs和4 977个新的lncRNAs,大部分lncRNAs呈低丰度表达。在两组小尾寒羊中发现120个差异表达lncRNAs,其中68个lncRNAs在高泌乳性能小尾寒羊中上调表达,52个lncRNAs下调表达。差异表达lncRNAs的靶基因显著富集在硫化物代谢过程、硫酯生物合成过程、酰基辅酶A生物合成过程、Rap1信号通路、粘附连接等通路上。LncRNA-miRNA网络分析发现,MSTRG.125242.6、MSTRG.59580.8等6个最显著差异表达lncRNAs的靶向miRNAs海绵体在家畜乳腺发育和泌乳过程中发挥了重要作用。RT-qPCR结果表明,16个lncRNAs的表达趋势与RNA-Seq结果完全吻合,证实了RNA-Seq测序结果的准确性和真实性。【结论】筛选的差异表达lncRNAs参与了绵羊乳腺发育及泌乳性能的调控,该结果将为解析绵羊泌乳性状的分子遗传机制提供理论参考。
王继卿,郝志云,沈继源,柯娜,黄兆春,梁维炜,罗玉柱,胡江,刘秀,李少斌. 小尾寒羊泌乳性状重要lncRNAs的筛选、鉴定及功能分析[J]. 中国农业科学, 2021, 54(14): 3113-3123.
WANG JiQing,HAO ZhiYun,SHEN JiYuan,KE Na,HUANG ZhaoChun,LIANG WeiWei,LUO YuZhu,HU Jiang,LIU Xiu,LI ShaoBin. Screening, Identification and Functional Analysis of Important LncRNAs for Lactation Traits in Small-Tailed Han Sheep[J]. Scientia Agricultura Sinica, 2021, 54(14): 3113-3123.
表1
RT-qPCR引物信息"
引物名称 Name | 正向序列(5'→3') Forward sequence (5'→3') | 反向序列(5'→3') Reverse sequence (5'→3') |
---|---|---|
MSTRG.59580.8 | ACCAGGTTGCCTAAGGAGGC | TGGAGTACAGTGGCTATTCACAA |
MSTRG.59580.9 | ACCAGGTTGCCTAAGGAGG | TGGAGTACAGTGGCTATTCACA |
MSTRG.59580.10 | TCGGGTGTCCGCACTAAG | GTTGCCCAGGCTGGAGTA |
MSTRG.38680.1 | GGTCTGCCTTCTTGGGTTC | TGGAGATGGAGCAGGGATC |
MSTRG.47064.1 | GCCGACTAAGGTCCATCT | GGCACTCAGCCTTCTTCA |
MSTRG.137650.1 | GTGCCGAAGAATAGATGC | TTCCTCCAAAGAAATCCC |
MSTRG.59580.12 | ACCAGGTTGCCTAAGGAGG | TGGAGTACAGTGGCTATTCACA |
MSTRG.80056.21 | CACTTCACTTAGCACGAT | CAACAGATAAATGGACGA |
MSTRG.125242.6 | GCACGAACAGCGACATCAG | CAGGGTCTCAGCAAGTTAGGAG |
MSTRG.119809.14 | TGTAGTCCTTTCCCAGTT | TGAATACCAGACCACCTTA |
MSTRG.59580.14 | GGCTGGAGGATCGCTTGA | TTGACCTGCTCCGTTTCC |
MSTRG.59580.11 | CAGCCTGGGCAACATAGC | CCGAACTTAGTGCGGACAC |
MSTRG.114625.4 | TGATCGCCAGGGTTGATT | GGATGGTCGTCCTCTTCG |
MSTRG.59580.3 | ACCAGGTTGCCTAAGGAGG | TGGAGTACAGTGGCTATTCACA |
MSTRG.106261.2 | TGTCACCCTCCACCAATG | CAGGCTGAAGTCCCAAAA |
rna-XR_003586216.1 | TACACGGTATTTCCTCCAA | CCAATTCTAAGATGCGATT |
GAPDH | ATCTCGCTCCTGGAAGATG | TCGGAGTGAACGGATTCG |
[1] | 中国畜禽遗传资源志: 羊志. 第一版. 北京: 中国农业出版社, 2011: 62-63. |
Animal Genetic Resources in China: Sheep and Goats. First edition. Beijing: China Agriculture Press, 2011: 62-63. (in Chinese) | |
[2] |
HIGHT G K, JURY K E. Hill country sheep production. II. Lamb mortality and birth weights in Romney and Border Leicester × Romney flocks. New Zealand Journal of Agricultural Research, 1970, 13:735-752.
doi: 10.1080/00288233.1970.10430507 |
[3] | 李庆章. 奶牛乳腺发育与泌乳生物学. 第一版. 北京: 科学出版社, 2014: 11-24. |
LI Q Z. Mammary gland development and lactation biology in dairy cows. First edition. Beijing: Science Press, 2014: 11-24. (in Chinese) | |
[4] |
GUTTMAN M, RINN J L. Modular regulatory principles of large non-coding RNAs. Nature, 2012, 482(7385):339-346.
doi: 10.1038/nature10887 |
[5] | 于红. 表观遗传学:生物细胞非编码RNA调控的研究进展. 遗传, 2009, 31(11):1077-1086. |
YU H. Epigenetics: Research progress in non-coding RNA regulation of biological cells. Heredity (Beijing), 2009, 31(11):1077-1086. (In Chinese) | |
[6] |
WILUSZ J E, SUNWOO H, SPECTOR D L. Long noncoding RNAs: functional surprises from the RNA world. Genes & Development, 2009, 23(13):1494-1504.
doi: 10.1101/gad.1800909 |
[7] |
IBEAGHA-AWEMU E M, LI R, DUDEMAINE P L, DO D N, BISSONNETTE N. Transcriptome analysis of long non-coding RNA in the bovine mammary gland following dietary supplementation with linseed oil and safflower oil. International Journal of Molecular Sciences, 2018, 19(11):3610.
doi: 10.3390/ijms19113610 |
[8] |
KNOWLING S, MORRIS K V. Non-coding RNA and antisense RNA. Nature’s trash or treasure? Biochimie, 2011, 93(11):1922-1927.
doi: 10.1016/j.biochi.2011.07.031 |
[9] |
CESANA M, CACCHIARELLI D, LEGNINI I, SANTINI T, STHANDIER O, CHINAPPI M, TRAMONTANO A, BOZZONI I. A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA. Cell, 2011, 147(2):358-369.
doi: 10.1016/j.cell.2011.09.028 |
[10] | CAO J. The functional role of long non-coding RNAs and epigenetics. Biological Procedures Online, 2014, 16:11. |
[11] |
STANDAERT L, ADRIAENS C, RADAELLI E, VAN KEYMEULEN A, BLANPAIN C, HIROSE T. The long noncoding RNA neat1 is required for mammary gland development and lactation. RNA, 2014, 20(12):1844-1849.
doi: 10.1261/rna.047332.114 |
[12] | GINGER M R, SHORE A N, CONTRERAS A, RIJNKELS M, MILLER J, GONZALEZ-RIMBAU M F, ROSEN J M. A noncoding RNA is a potential marker of cell fate during mammary gland development. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(15):5781-5786. |
[13] |
LANZ R B, CHUA S S, BARRON N, SÖDER B M, DEMAYO F, O'MALLEY B W. Steroid receptor RNA activator stimulates proliferation as well as apoptosis in vivo. Molecular and Cellular Biology, 2003, 23(20):7163-7176.
doi: 10.1128/MCB.23.20.7163-7176.2003 |
[14] |
YANG B, JIAO B, GE W, ZHANG X, WANG S, ZHAO H, WANG X. Transcriptome sequencing to detect the potential role of long non-coding RNAs in bovine mammary gland during the dry and lactation period. BMC Genomics, 2018, 19(1):605.
doi: 10.1186/s12864-018-4974-5 |
[15] |
ZHENG X, NING C, ZHAO P, FENG W, JIN Y, ZHOU L, YU Y, LIU J. Integrated analysis of long noncoding RNA and mRNA expression profiles reveals the potential role of long noncoding RNA in different bovine lactation stages. Journal of Dairy Science, 2018, 101(12):11061-11073.
doi: 10.3168/jds.2018-14900 |
[16] |
YU S, ZHAO Y, LAI F, CHU M, HAO Y, FENG Y, ZHANG H, LIU J, CHENG M, LI L, SHEN W, MIN L. LncRNA as ceRNAs may be involved in lactation process. Oncotarget, 2017, 8(58):98014-98028.
doi: 10.18632/oncotarget.v8i58 |
[17] |
JI Z, CHAO T, LIU Z, HOU L, WANG J, WANG A, ZHOU J, XUAN R, WANG G, WANG J. Genome-wide integrated analysis demonstrates widespread functions of lncRNAs in mammary gland development and lactation in dairy goats. BMC Genomics, 2020, 21(1):254.
doi: 10.1186/s12864-020-6656-3 |
[18] |
NI Y, WU F, CHEN Q, CAI J, HU J, SHEN J, ZHANG J. Long noncoding RNA and mRNA profiling of hypothalamic- pituitary-mammary gland axis in lactating sows under heat stress. Genomics, 2020, 112(5):3668-3676.
doi: 10.1016/j.ygeno.2020.04.021 |
[19] |
CHEN W, LV X, WANG Y, ZHANG X, WANG S, HUSSAIN Z, CHEN L, SU R, SUN W. Transcriptional profiles of long non-coding RNA and mRNA in sheep mammary gland during lactation period. Frontiers in Genetics, 2020, 11:946.
doi: 10.3389/fgene.2020.00946 |
[20] |
HAO Z, LUO Y, WANG J, HU J, LIU X, LI S, JIN X, KE N, ZHAO M, HU L, WU X, QIAO L. RNA-Seq reveals the expression profiles of long non-coding RNAs in lactating mammary gland from two sheep breeds with divergent milk phenotype. Animals, 2020, 10(9):1565.
doi: 10.3390/ani10091565 |
[21] | 赵有璋. 中国养羊学. 北京: 中国农业出版社, 2013: 666. |
ZHAO Y Z. Sheep and goat production in China. Beijing: China Agriculture Press, 2013: 666. (in Chinese) | |
[22] | 韦科龙, 谭正准, 黄健, 李辉, 钟华配, 覃广胜. 奶水牛乳腺组织手术采样. 黑龙江畜牧兽医, 2017, 4:97-98. |
WEI K L, TAN Z Z, HUANG J, LI H, ZHONG H P, QIN G S. Surgical sampling of mammary gland tissue of milk buffalo. Heilongjiang Animal Science and Veterinary Medicine, 2017, 4:97-98. (In Chinese) | |
[23] | 杨兵. 奶牛乳腺差异表达长链非编码RNA的筛选, 鉴定及其功能研究[D]. 杨凌: 西北农林科技大学, 2019. |
YANG B. Screening, identification and functional studies of long non-coding RNAs differentially expressed in mammary gland of dairy cows[D]. Yangling: Northwest A&F University, 2019. (in Chinese) | |
[24] |
AKERS R M. A 100-year review: Mammary development and lactation. Journal of Dairy Science, 2017, 100(12):10332-10352.
doi: 10.3168/jds.2017-12983 |
[25] |
LI Z, HUANG C, BAO C, CHEN L, LIN M, WANG X, ZHONG G, YU B, HU W, DAI L, ZHU P, CHANG Z, WU Q, ZHAO Y, JIA Y, XU P, LIU H, SHAN G. Exon-intron circular RNAs regulate transcription in the nucleus. Nature Structural & Molecular Biology, 2015, 22(3):256-264.
doi: 10.1038/nsmb.2959 |
[26] |
LAHIRY P, WANG J, ROBINSON J F, TUROWEC J P, LITCHFIELD D W, LANKTREE M B, GLOOR G B, PUFFENBERGER E G, STRAUSS K A, MARTENS M B, RAMSAY D A, RUPAR C A, SIU V, HEGELE R A. A multiplex human syndrome implicates a key role for intestinal cell kinase in development of central nervous, skeletal, and endocrine systems. American Journal of Human Genetics, 2009, 84(2):134-147.
doi: 10.1016/j.ajhg.2008.12.017 |
[27] |
VERARDO L L, SILVA F F, VARONA L, RESENDE M D, BASTIAANSEN J W, LOPES P S, GUIMARÃES S E. Bayesian GWAS and network analysis revealed new candidate genes for number of teats in pigs. Journal of Applied Genetics, 2015, 56(1):123-132.
doi: 10.1007/s13353-014-0240-y |
[28] |
WINTERMANTEL T M, BOCK D, FLEIG V, GREINER E F, SCHÜTZ G. The epithelial glucocorticoid receptor is required for the normal timing of cell proliferation during mammary lobuloalveolar development but is dispensable for milk production. Molecular Endocrinology, 2019, 19(2):340-349.
doi: 10.1210/me.2004-0068 |
[29] |
Cowell I G. E4BP4/NFIL3, a PAR-related bZIP factor with many roles. BioEssays, 2002, 24(11):1023-1029.
pmid: 12386933 |
[30] |
GHADIRI S, SPALENZA V, DELLAFIORA L, BADINO P, BARBAROSSA A, DALL'ASTA C, NEBBIA C, GIROLAMI F. Modulation of aflatoxin B1 cytotoxicity and aflatoxin M1 synthesis by natural antioxidants in a bovine mammary epithelial cell line. Toxicology in vitro, 2019, 57:174-183.
doi: 10.1016/j.tiv.2019.03.002 |
[31] |
AHN J, GAMMON M D, SANTELLA R M, GAUDET M M, BRITTON J A, TEITELBAUM S L, TERRY M B, NEUGUT A I, ENG S M, ZHANG Y, GARZA C, AMBROSONE C B. Effects of glutathione S-transferase A1 (GSTA1) genotype and potential modifiers on breast cancer risk. Carcinogenesis, 2006, 27(9):1876-1882.
doi: 10.1093/carcin/bgl038 |
[32] |
SHAO S, BROWN A, SANTHANAM B, HEGDE R S. Structure and assembly pathway of the ribosome quality control complex. Molecular Cell, 2015, 57(3):433-444.
doi: 10.1016/j.molcel.2014.12.015 |
[33] |
BI X, JONES T, ABBASI F, LEE H, STULTZ B, HURSH D A, MORTIN MA. Drosophila caliban, a nuclear export mediator, can function as a tumor suppressor in human lung cancer cells. Oncogene, 2005, 24(56):8229-8239.
doi: 10.1038/sj.onc.1208962 |
[34] |
HUANG Z, PANG G, HUANG Y G, LI LI. miR-133 inhibits proliferation and promotes apoptosis by targeting LASP1 in lupus nephritis. Experimental and Molecular Pathology, 2020, 114:104384.
doi: 10.1016/j.yexmp.2020.104384 |
[35] | GÜRTLER F, JORDAN K, TEGTMEIER I, HEROLD J, STINDL J, WARTH R, BANDULIK S. Cellular pathophysiology of mutant voltage-dependent Ca2+ channel CACNA1H in primary aldosteronism. Endocrinology, 2020, 161(10):135. |
[36] | REINHARDT T A, HORST R L. Ca2+-ATPases and their expression in the mammary gland of pregnant and lactating rats. American Journal of Physiology, 1999, 276(4):796-802. |
[37] |
CHEN Z, LUO J, SUN S, CAO D, SHI H, LOOR J J. miR-148a and miR-17-5p synergistically regulate milk TAG synthesis via PPARGC1A and PPARA in goat mammary epithelial cells. RNA Biology, 2017, 14(3):326-338.
doi: 10.1080/15476286.2016.1276149 |
[38] | 黄千殷, 曹小迎, 张宇琛, 刘天罡, 徐焱成. 通过硫酯反应检测脂肪酸代谢中间产物. 武汉大学学报 (医学版), 2017, 38(5):724-727. |
HUANG Q Y, CAO X Y, ZHANG Y C, LIU T G, XU Y C. Application of a novel thioester decomposition reaction in fatty acid biosynthesis intermediates analysis. Medical Journal of Wuhan University, 2017, 38(5):724-727. (in Chinese) | |
[39] |
HOU X, JIANG M, ZHOU J, SONG S, ZHAO F, LIN Y. Examination of methionine stimulation of gene expression in dairy cow mammary epithelial cells using RNA-sequencing. Journal of Dairy Research, 2020, 87(2):226-231.
doi: 10.1017/S0022029920000199 |
[40] |
FORNETTI J, FLANDERS K C, HENSON P M, TAN A C, BORGES V F, SCHEDIN P. Mammary epithelial cell phagocytosis downstream of TGF-β3 is characterized by adherens junction reorganization. Cell Death and Differentiation, 2016, 23(2):185-196.
doi: 10.1038/cdd.2015.82 |
[1] | 董复成,马淑丽,时娟娟,张俊梅,崔岩,任有蛇,张春香. LCN5蛋白在雄性绵羊生殖器官及精子中的表达定位[J]. 中国农业科学, 2022, 55(7): 1445-1457. |
[2] | 梁鹏,张天闻,孟科,邵顺成,邹诗凡,荣轩,强浩,冯登侦. 绵羊ADIPOQ多态性与生长性状的关联分析[J]. 中国农业科学, 2022, 55(11): 2239-2256. |
[3] | 柯娜,郝志云,王建清,甄慧敏,罗玉柱,胡江,刘秀,李少斌,赵志东,黄兆春,梁维炜,王继卿. miR-221靶向IRS1抑制绵羊乳腺上皮细胞活力和增殖[J]. 中国农业科学, 2022, 55(10): 2047-2056. |
[4] | 姜春晖,孙旭东,唐燕,罗胜缤,徐闯,陈媛媛. 姜黄素通过Nrf2信号通路对H2O2诱导奶牛乳腺上皮细胞氧化应激的缓解[J]. 中国农业科学, 2021, 54(8): 1787-1794. |
[5] | 李松美,仇雨歌,陈胜男,王晓萌,王春生. CRISPR/Cas9介导的绵羊示踪脐带间充质干细胞系的建立[J]. 中国农业科学, 2021, 54(2): 400-411. |
[6] | 陈志,张逸,路钦越,郭佳禾,梁艳,张明怡星,杨章平. 茶树油对LPS诱导的奶牛乳腺炎的作用及其机制[J]. 中国农业科学, 2021, 54(14): 3124-3133. |
[7] | 宋美洁,欧爱群,薛晓锋,吴黎明,寿旗扬,王凯. 蜂胶提取物对脂多糖诱导小鼠急性乳腺炎及乳腺屏障功能的保护作用[J]. 中国农业科学, 2021, 54(12): 2675-2688. |
[8] | 石田培,王欣悦,侯浩宾,赵志达,尚明玉,张莉. 基于全转录组测序的绵羊胚胎不同发育阶段 骨骼肌circRNA的分析与鉴定[J]. 中国农业科学, 2020, 53(3): 642-657. |
[9] | 李讨讨,王霞,马友记,尹德恩,张勇,赵兴绪. 藏绵羊BOLL的分子特征及其在睾丸中的表达调控与功能分析[J]. 中国农业科学, 2020, 53(20): 4297-4312. |
[10] | 郑玮才,郝小燕,张宏祥,项斌伟,张文佳,张春香,张建新. 饲粮添加酿酒酵母和地衣芽孢杆菌对绵羊生长性能与瘤胃发酵的影响[J]. 中国农业科学, 2020, 53(16): 3385-3393. |
[11] | 王欣悦,石田培,赵志达,胡文萍,尚明玉,张莉. 基于绵羊胚胎骨骼肌蛋白质组学的PI3K-AKT信号通路分析[J]. 中国农业科学, 2020, 53(14): 2956-5963. |
[12] | 田志龙,汤继顺,孙庆,王玉琴,张效生,张金龙,储明星. 绵羊SMAD1基因组织表达及其多态性与产羔数关联分析[J]. 中国农业科学, 2019, 52(4): 755-766. |
[13] | 富丽霞,马涛,刁其玉,成述儒,宋雅喆,孙卓琳. 肉羊精料可代谢蛋白质预测模型的建立[J]. 中国农业科学, 2019, 52(3): 539-549. |
[14] | 王丽芳,张兴夫. 黄花蒿醇提物对奶牛乳腺细胞中共轭亚油酸 合成相关酶基因表达的作用[J]. 中国农业科学, 2019, 52(18): 3271-3278. |
[15] | 毕崇亮,刘俊俊,王亨,王娟,韩照清,关立增. 硒对S. aureus诱导的奶牛乳腺上皮细胞Nod2/MAPK/mTORs信号通路关键蛋白表达的影响[J]. 中国农业科学, 2019, 52(16): 2891-2898. |
|