营养诱导舍饲哈萨克羊非繁殖季节卵巢组织中miRNAs差异表达分析

doi:10.11843/j.issn.0366-6964.2017.04.005

摘要/Abstract

摘要：

旨在利用Solexa测序技术分析、挖掘营养诱导非繁殖季节绵羊发情期和乏情期卵巢组织micro RNAs（miRNA），为进一步研究特定miRNA参与非繁殖季节绵羊发情的分子调控机制提供理论基础。以不同营养水平饲喂的哈萨克绵羊作为研究对象，在非繁殖季节前后分别屠宰已鉴定的3只发情（EN）和乏情（AN）母羊，分别采集卵巢（O）组织，构建两个小片段RNA文库（OEN和OAN），进行高通量测序和生物信息学分析，实时荧光定量PCR（qRT-PCR）验证差异表达的miRNAs在哈萨克羊卵巢中表达水平。结果显示，两文库分别获得8 665 978（发情期）和9 071 102条（乏情期）clean reads，且绝大多数小RNA序列长度为21~23 nt。两文库相比较，发现有9个已知和104个未知miRNAs表达差异显著（P<0.01）。利用qRT-PCR随机选择的6个显著差异表达的miRNAs进行验证，其表达水平和测序分析结果一致。KEGG通路分析表明，这些差异miRNAs的靶基因主要参与了甘氨酸、丝氨酸和苏氨酸代谢，氨基糖和核苷酸糖代谢，精氨酸和鸟氨酸代谢及甘油磷脂代谢等营养调控通路。结合靶基因预测及通路富集分析，推测差异表达的miRNAs是通过营养代谢通路对绵羊的非季节性发情起重要作用。

关键词: 哈萨克羊, 发情期, 乏情期, 卵巢, Solexa测序, microRNA

Abstract:

To further explore the theoretical basis of the microRNAs in regulating the non-breeding season estrous in sheep, characterization of ovarian microRNAs were constructed and analyzed in estrous and anestrous of non-breeding season induced by nutrition by Solexa sequencing. In this study, the Kazakh sheep with different nutrition levels were used as the research object. In non-breeding season,3 estrous (EN) and 3 anestrous (AN) sheep were slaughtered with their ovary tissue(O) collected, respectively. Two miRNA libraries were constructed and analyzed by the bioinformatics methods. The real-time quantitative PCR (qRT-PCR) was used for validating the differentially expressed miRNAs betweent the 2 periods in ovary. The results showed that the clean reads of 8 665 978 (OEN) and 9 071 102 (OAN) were obtained in the two libraries, and most of the small RNA sequences were between 21 and 23 nt. Compared the two ovary (OEN vs OAN) libaries, the 9 known and 104 novel miRNAs had significant expression difference (P<0.01). The expression level of 6 randomly selected miRNAs by qRT-PCR was the same as the result by Solexa sequencing. KEGG pathway analysis showed that these target genes of difference miRNAs were primarily involved in nutrition regulatory related pathways, such as glycine, serine and threonine metabolism, amino sugar and nucleotide sugar metabolism, D-arginine and D-ornithine metabolism, glycerol phospholipid metabolism, and so on. The differentially expressed miRNAs may play an important role in non-breeding season estrous of sheep through nutrition metabolism pathway by the analysis of predicted target and enriched pathway analysis.

Key words: Kazakh sheep, estrus, anestrus, ovary, solexa sequencing, microRNA

中图分类号:

S826.8⁺4.2

雷小萍, 林杉, 于要升, 梁慧慧, 赵宗胜. 营养诱导舍饲哈萨克羊非繁殖季节卵巢组织中miRNAs差异表达分析[J]. 畜牧兽医学报, 2017, 48(4): 627-636.

LEI Xiao-ping, LIN Shan, YU Yao-sheng, LIANG Hui-hui, ZHAO Zong-sheng. Analyzing the Differential Expression of miRNAs in Ovary of Stall-feed Kazakh Sheep Induced by Nutrition in Non-breeding Season[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(4): 627-636.

参考文献

[1] BARTEL D P. microRNAs:genomics, biogenesis, mechanism, and function[J]. Cell, 2004, 116(2):281-297.
[2] ECKSTEIN F.Small non-coding RNAs as magic bullets[J].Trends Biochem Sci, 2005, 30(8):445-452.
[3] SHENG Y R, LI J B, ZOU C C, et al. Down regulation of miR-101-3p by hepatitis B virus promotes proliferation and migration of hepatocellular carcinoma cells by targeting Rab5a[J]. Arch Virol, 2014, 159(9):2397-2410.
[4] 徐源,孙铁成,张爱玲,等.卵巢miRNA研究进展[J].畜牧兽医学报,2014,45(4):509-516.
XU Y,SUN T C,ZHANG A L,et al.Progress on the research of ovarian miRNA[J]. Acta Veterinaria et Zootechnica Sinica, 2014, 45(4):509-516. (in Chinese)
[5] HAYASHI K,DE SOUSA LOPES S M C,KANEDA M,et al.microRNA biogenesis is required for mouse primordial germ cell development and spermatogenesis[J]. PLoS One, 2008, 3(3):1738.
[6] MEDEIROS L A,DENNIS L M,GILL M E,et al.Mir-290-295 deficiency in mice results in partially penetrant embryonic lethality and germ cell defects[J].Proc Natl Acad Sci USA, 2011, 108(34):14163-14168.
[7] XU S,LINHER-MELVILLE K,YANG B B,et al.micro-RNA378(miR-378) regulates ovarian estradiol production by targeting aromatase[J]. Endocrinology, 2011, 152(10):3941-3951.
[8] DI R,HE J N, SONU S H, et al. Characterization and comparative profiling of ovarian microRNAs during ovine anestrus and the breeding season[J]. BMC Genomics, 2014, 15:899.
[9] 黄冬维,储明星.动物季节性繁殖分子调控机理研究进展[J].遗传, 2011, 33(7):35-46.
HUANG D W, CHU M X.Research progress in molecular mechanism of animal seasonal reproduction[J]. Hereditas (Beijing), 2011, 33(7):35-46. (in Chinese)
[10] KIYMA Z, ALEXANDER B M, VAN KIRE E A, et al. Effects of feed restriction on reproductive and metabolic hormones in ewes[J]. J Anim Sci, 2004, 82(9):2548-2557.
[11] 马辽伟,侯生珍,王志有,等.补饲不同能量水平的精料补充料对放牧藏母羊发情受胎的影响[J]. 江苏农业科学, 2015, 43(12):242-244.
MA L W, HOU S Z, WANG Z Y,et al. Effects of supplementary concentrate supplemented with different energy levels on embryo conception of sheep[J]. Jiangsu Agricultural Sciences, 2015, 43(12):242-244. (in Chinese)
[12] 姚洪军,赵春雨,仲昭鑫,等.添加复合营养补充剂对奶牛发情及产奶性能的影响[J].中国兽医杂志,2016,52(10):14-16.
YAO H J, ZHAO C Y, ZHONG Z X, et al. Effects of compound nutritional supplement on estrus and milk production of dairy cows[J]. Chinese Journal of Veterinary Medicine, 2016, 52(10):14-16. (in Chinese)
[13] VIÑOLES C, FORSBERG M, MARTIN G B, et al. Short-term nutritional supplementation of ewes in low body condition affects follicle development due to an increase in glucose and metabolic hormones[J]. Reproduction, 2005, 129(3):299-309.
[14] SHORT R E,ADAMS D C.Nutritional and hormonal interrelationships in beef cattle reproduction[J]. J Anim Sci, 1998, 68(1):29-39.
[15] 景炜,蒲松.日粮不同能量和蛋白水平对多浪羊繁殖性能及血液生化指标的影响[J]. 中国草食动物, 2010, 30(5):21-25.
JING W, PU S. Effect of diets with different level energy and protein on reproductive performance and serum biochemical indexes in ewes[J]. China Herbivore, 2010, 30(5):21-25. (in Chinese)
[16] AKDHAVA A P,MEHER A A,JOSHI S R.Preconceptional omega-3 fatty acid supplementation on a micronutrient-deficient diet improves the reproductive cycle in Wistar rats[J].Reprod Fertil Dev, 2013, 25(7):1085-1094.
[17] 刘顺德,时兴伟,杨涛. 能量和蛋白质摄入量对母猪繁殖性能的影响[J].畜牧兽医杂志, 2001, 20(3):25-29.
LIU S D, SHI X W, YANG T. Effect of Energy and Protein Intake on the Reproductive Performance of Sow[J]. Journal of Animal Science and Veterinary Medicine, 2001, 20(3):25-29.(in Chinese)
[18] AN X P, SONG Y X, HOU J X, et al. Identification and profiling of microRNAs in the ovaries of polytocous and monotocous goats during estrus[J]. Theriogenology, 2016, 85(4):769-780.
[19] JOHNSON A E M, FREEMAN E W, COLGIN M, et al. Induction of ovarian activity and ovulation in an induced ovulator, the maned wolf (Chrysocyon brachyurus), using GnRH agonist and recombinant LH[J]. Theriogenology, 2014, 82(1):71-79.
[20] GE L C,CHEN Z J,LIU H Y,et al.Involvement of activating ERK1/2 through G protein coupled receptor 30 and estrogen receptor α/β in low doses of bisphenol A promoting growth of Sertoli TM4 cells[J]. Toxicol Lett, 2014, 226(1-2):81-89.
[21] PANGAS S A. Bone morphogenetic protein signaling transcription factor (SMAD) function in granulosa cells[J]. Mol Cell Endocrinol,2012, 356(1):40-47.
[22] 段新崇,魏彦辉,李阳,等.小尾寒羊间情期和发情期microRNAs差异表达分析[J].畜牧兽医学报, 2016, 47(7):1324-1332.
DUAN X C,WEI Y H,LI Y,et al.Analyzing the differential expression of microRNAs in estrus and diestrus of Small Tailed Han sheep[J]. Acta Veterinaria et Zootechnica Sinica, 2016, 47(7):1324-1332. (in Chinese)
[23] POGRIBNY I P,TYRNDYAK V P,ROSS S A,et al.Differential expression of microRNAs during hepatocarcinogenesis induced by methyl deficiency in rats[J].Nutr Rev,2008, 66(S1):S33-S35.
[24] ZHANG J,ZHANG F,DIDEL O T,et al.Maternal high fat diet during pregnancy and lactation alters hepatic expression of insulin like growth factor-2 and key microRNAs in the adult offspring[J]. BMC Genomics, 2009, 10:478.
[25] BEAGLE B,YANG T L,HUNG J,et al.The glycine N-methyltransferase (GNMT) 1289 C->T variant influences plasma total homocysteine concentrations in young women after restricting folate intake[J]. J Nutr, 2005, 135(12):2780-2785.
[26] 安宇.叶酸代谢网络酶基因遗传多态性与原发性肝癌的遗传易感性的研究[D].上海:复旦大学,2008.
AN Y.Association study on polymorphisms of one-carbon pathway with susceptibility of Hepatocellular carcinoma[D]. Shanghai:Fudan University, 2008. (in Chinese)
[27] 高振华,张军,孟艳,等.叶酸水平对母猪繁殖性能的影响[J].饲料研究,2010(12):18-20.
GAO Z H, ZHANG J, MENG Y, et al. Effects of folic acid level on reproductive performance of sow[J]. Feed Research, 2010(12):18-20. (in Chinese)
[28] LUO F, JIA R, YING S, et al. Analysis of genes that influence sheep follicular development by different nutrition levels during the luteal phase using expression profiling[J]. Anim Genet, 2016, 47(3):354-364.
[29] MEHRABIAN M, SCHULTHESS F T, NEBOHACOVA M, et al. Identification of ALOX5 as a gene regulating adiposity and pancreatic function[J]. Diabetologia, 2008, 51(6):978-988.
[30] 孔路军, 傅金恋, 刘宗慧, 等. 脂肪型和瘦肉型猪肥胖基因表达差异及其与繁殖性能的关系[J]. 中国畜牧杂志, 2005,41(7):9-12.
KONG L J, FU J L, LIU Z H, et al. Expression difference of obese gene between fat and lean type pig and correlation with reproduction performance[J]. Chinese Journal of Animal Science, 2005, 41(7):9-12. (in Chinese)
[31] 齐长学, 付世新, 张丽, 等. 瘦素(leptin)与雌性哺乳动物的繁殖活动关系的研究进展[J]. 中国奶牛, 2008(10):38-40.
QI C X, FU S X, ZHANG L, et al. Research advance in the relationship between leptin and propagation of female mammalian[J]. China Dairy Cattle, 2008(10):38-40. (in Chinese)