绵羊尾脂沉积相关circRNA的鉴定及功能验证

doi:10.7668/hbnxb.20195101

摘要/Abstract

摘要：

绵羊的尾型是由遗传和环境因素相互作用形成的复杂性状,环状RNA(circRNA)与脂肪生成密切相关。为探究circRNA对绵羊尾部脂肪沉积的影响,对绵羊尾部脂肪进行转录组测序并进行样品组间差异表达分析,筛选出与绵羊尾脂相关的候选circRNA,构建绵羊尾部脂肪沉积相关circRNA-miRNA-mRNA 的调控网络图,并对筛选出来的circRNA进行定位,然后对其功能进行验证。结果显示,在2种不同尾型绵羊尾部脂肪组织转录本中共筛选得到679个差异表达circRNA,其中422个上调,257个下调。并对差异circRNA靶基因进行GO和KEGG功能富集分析,涉及了DNA代谢过程、解剖结构发育、分解代谢过程、细胞自噬作用、碳水化合物吸收过程以及脂肪沉积相关的细胞增殖、脂质代谢等众多生物学发育过程。靶基因富集在细胞生长与凋亡过程、细胞运动、运输和分解代谢、信号转导作用、转录翻译以及氨基酸合成代谢等功能,说明这些circRNA可能通过以上众多通路参与绵羊尾部脂肪的沉积过程。对筛选出的差异circRNA_0018采用荧光原位杂交方法进行定位分析并在前体脂肪细胞中进行验证,结果表明,circRNA_0018是真正且稳定的细胞质环状分子,能促进脂肪细胞分化,由此推测,circRNA_0018等可能参与绵羊脂肪沉积和脂质代谢过程。

关键词: 绵羊, 基因表达, 功能验证, 尾脂沉积, circRNA

Abstract:

The tail type of sheep is a complex trait formed by the interaction of genetic and environmental factors.circRNA is closely related to lipogenesis.To investigate the effect of circular RNA(circRNA)on the tail fat deposition of sheep,transcriptome sequencing and differential expression analysis of sheep tail fat were performed.Candidate circRNA associated with sheep tail fat were screened,and the regulatory network diagram of circRNA-miRNA-mRNA associated with sheep tail fat deposition was constructed,the selected circRNA were located,and their functions were verified.The results showed that a total of 679 differentially expressed circRNA were detected in the transcripts of adipose tissue of two different tail types of sheep,of which 422 were up-regulated and 257 down-regulated.Moreover,GO and KEGG functional enrichment analysis was performed on differentially circRNA target genes,which involved many biological development processes such as DNA metabolism,anatomical structure development,catabolic process,autophagy,carbohydrate absorption process,cell proliferation and lipid metabolism related to fat deposition.Target gene enrichment was involved in cell growth and apoptosis,cell motility,transport and catabolism,signal transduction,transcription and translation,amino acid anabolism and other functions,suggesting that these circRNA may participate in the deposition process of sheep tail fat through the above pathways.The selected differential circRNA_0018 was localized by fluorescence in situ hybridization and verified in the precursor adipocytes.The results showed that circRNA_0018 was a true and stable cytoplasmic ring molecule,and functional verification of circRNA_0018 showed that it could promote adipocyte differentiation.circRNA_0018 may be involved in the process of fat deposition and lipid metabolism in sheep.

Key words: Sheep, Gene expression, Functional verification, Tail lipid deposition, circRNA

牛瑞来, 张悦, 韦应实, 杨飏, 卿宇, 成述儒, 朱才业. 绵羊尾脂沉积相关circRNA的鉴定及功能验证[J]. 华北农学报, 2025, 40(2): 191-200. doi: 10.7668/hbnxb.20195101.

NIU Ruilai, ZHANG Yue, WEI Yingshi, YANG Yang, QING Yu, CHENG Shuru, ZHU Caiye. Identification and Functional Analysis of circRNA Associated with Tail Fat Deposition in Sheep[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(2): 191-200. doi: 10.7668/hbnxb.20195101.

http://www.hbnxb.net/CN/Y2025/V40/I2/191

图/表 10

图1 总RNA凝胶电泳 1—3.阿勒泰羊的3个样品;4—6.藏羊的3个样品;M.Marker Trans 2K Plus。

Fig.1 Total RNA gel electrophoresis 1—3.Three samples of Altay sheep;4—6.Three samples of Tibetan sheep; M.Marker Trans 2K Plus.

表1 过滤前后reads及碱基信息统计

Tab.1 Statistical of reads and bases in formation before and after filtration

样品 Sample		AL1
过滤前	Raw reads	84 131 872	91 269 246	84 939 878	80 392 582	85 561 024	80 392 582
Before filter	Clean reads	12 619 780 800	13 690 386 900	12 740 981 700	12 058 887 300	12 834 153 600	12 058 887 300
	Q20/%	97.96	97.97	96.79	97.71	98.05	97.71
	Q30/%	94.60	94.65	92.14	93.97	94.76	93.97
	GC/%	50.70	48.01	51.22	53.63	50.26	53.63
过滤后	Raw reads	83 218 514	89 585 054	83 947 804	79 416 444	84 363 650	90 813 024
After filter	Clean reads	12 482 777 100	13 437 758 100	12 592 170 600	11 912 466 600	12 654 547 500	11 912 466 600
	Q20/%	97.97	97.96	92.17	97.71	98.05	98.02
	Q30/%	94.60	94.63	96.83	93.96	94.75	94.72
	GC/%	50.71	47.96	51.20	53.60	50.20	50.74

图2 差异表达基因表达量密度分布图(A)和箱线图(B) A.横坐标为表达量(TPM值)取以10为底的对数,纵坐标轴为不同表达量数值的基因的密度,不同颜色代表不同样品。B.横坐标为样品名称,纵坐标为log₁₀(TPM值),每个区域的箱线图对应5个统计量。

Fig.2 Expression density distribution of differentially expressed genes(A)and boxplot(B) A.The horizontal coordinate is the logarithm of the expression quantity(TPM value)at the base of 10,the vertical axis is the density of genes with different expression quantity values,and different colors represent different samples.B.The horizontal coordinate is the sample name,the vertical coordinate is log₁₀(TPM value),and the five statistics corresponding to the box plot of each region.

图3 差异表达circRNA火山图(A)和差异表达circRNA聚类热图(B)

Fig.3 Differentially expressed circRNA volcano map(A)and differentially expressed circRNA clustering heat map(B)

图4 circRNA-miRNA-mRNA的ceRNA网络图

Fig.4 ceRNA network of circRNA-miRNA-mRNA

图5 差异表达基因的GO功能富集图点的颜色代表校正后的 P value,点的大小代表差异表达基因的数目。1.ADP结合;2.血红素结合;3.信息转导;4.转移酶活性,转移己糖基;5.跨膜转运蛋白活性;6.蛋白苏氨酸激酶活性;7.细胞壁大分子分解过程;8.纤维素生物合成工艺;9.磷脂酰肌醇代谢过程;10.氧化还原酶活性,作用于单个供体,结合分子氧,结合2个氧原子;11.磷脂酰肌醇硫酸激酶活性;12.对生物刺激的反应;13.细胞骨架组织;14.细胞色素c氧化酶活性;15.碱基切除修复;16.纤维素合成酶活性;17.蛋白插入膜;18.肽-蛋氨酸-s-氧化物还原酶活性;19.连接酶活性,形成氨基酰基-tRNA和相关化合物;20.转移酶活性,转移相关基团。

Fig.5 GO functional enrichment map of differentially expressed genes The color of the dots represents the corrected P value,and the size of the dots represents the number of differentially expressed genes.1.ADP binding; 2.Heme binding; 3.Signal transduction; 4.Transferase activity,transferring hexosyl groups; 5.Transmembrane transporter activity;6.Protein serine/threonine kinase activity; 7.Cell wall macromolecule catabolic process; 8.Cellulose biosynthetic process; 9.Phosphatidylinositol metabolic process; 10.Oxidoreductase activity,acting on single donors with incorporation of molecular oxygen,incorporation of two atoms of oxygen; 11. Phosphatidylinositol phosphate kinase activity; 12.Response to biotic stimulus; 13.Cytoskeleton organization; 14.Cytochrome-c oxidase activity; 15.Base-excision repair;16.Cellulose synthase(UDP-forming)activity; 17.Protein insertion into membrane; 18.Peptide-methionine(S)-S-oxide reductase activity; 19.Ligase activity,forming aminoacyl-tRNA and related compounds; 20.Transferase activity,transferring phosphorus-containing groups.

图6 差异表达基因的KEGG功能富集图纵轴为基因比例(该 Pathway 中的差异基因占所有差异基因的比例),横轴为 KEGG Pathway, 点的颜色代表校正后的 P value,点的大小代表差异表达基因的数目。1.苯丙素的生物合成;2.氨基酸合成;3.半胱氨酸和蛋氨酸代谢;4.2-氧羧酸代谢;5.药物代谢-细胞色素P450;6.细胞色素P450对外源性药物的代谢作用;7.化学致癌作用;8.缬氨酸、亮氨酸和异亮氨酸的降解;9.氰氨酸代谢;10.色氨酸代谢;11.L-亚麻酸代谢;12.半乳糖代谢;13.β-氨基酸代谢;14.缬氨酸、亮氨酸、异亮氨酸生物合成;15.芥子油甘生物合成;16.类黄酮生物合成;17.肥厚性心肌病(HCM);18.油菜素内酯生物合成;19.氯烷和氯烯烃降解;20.亚油酸代谢。

Fig.6 KEGG functional enrichment map of differentially expressed genes The vertical axis is Gene ratio(the proportion of differential genes in this Pathway to all differential genes), the horizontal axis is KEGG Pathway, the color of dots represents the corrected P value, and the size of dots represents the number of differentially expressed genes.1.Phenylpropanoid biosynthesis; 2.Biosynthesis of amino acids; 3.Cysteine and mehionine metatolism;4.2-Oxocarboxylc acid metabolism;5.Drug melabolism-cylochrome P450; 6.Metabolism of xenobiotics by cylochrome P450; 7.Chemical crcinogenesis; 8.Valine,leucine and isieucine degradation; 9.Cyanoamino acid metabolism; 10.Trypiophan metabolism; 11.alpha-Linolenic acid metabolism; 12.Galactose meiabollsm; 13.beta-Alanine metabolism; 14.Vallne,leucine and lsoleucine blosynthesis; 15.Glucosinolate biosynthesls; 16.Flavonold blosynthesis; 17.Hypertrophie cardiomyopathy(HCM); 18.Brassinosterold biosynthesis; 19.Chloroalkane and chioroalkene degradation; 20.Linoleic acid metabolism.

图7 circRNA_0018的RNA FISH

Fig.7 RNA FISH of circRNA_0018

图8 显微镜下油红O染色结果

Fig.8 Results of oil red O staining under the microscope

图9 RT-PCR检测细胞中LPL基因mRNA表达结果 M.Marker;1.pCAMBIA1305-circRNA表达载体;2.pCAMBIA1305空载体;3.对照组。

Fig.9 mRNA expression of LPL gene in cells detected by RT-PCR M.Marker; 1.pCAMBIA1305-circRNA expression vector;2.pCAMBIA1305 empty carrier; 3.Control group.

参考文献 32

[1]	国家畜禽遗传资源委员会组编. 中国畜禽遗传资源志—羊志[M]. 北京: 中国农业出版社, 2011.
	National Livestock and Poultry Genetic Resources Committee organized. Chinese livestock and poultry genetic resources-sheep[M]. Beijing: China Agriculture Press, 2011.
[2]	Ermias E, Yami A, Rege J E O. Fat deposition in tropical sheep as adaptive attribute to periodic feed fluctuation[J]. Journal of Animal Breeding and Genetics, 2002, 119(4): 235-246. doi: 10.1046/j.1439-0388.2002.00344.X
[3]	Kashan N E J, Manafi Azar G H, Afzalzadeh A, Salehi A. Growth performance and carcass quality of fattening lambs from fat-tailed and tailed sheep breeds[J]. Small Ruminant Research, 2005, 60(3):267-271.doi:10.16213/j.cnki.scjas.2013.05.075.
[4]	甘尚权, 张伟, 宋天增, 沈敏, 梁耀伟, 杨井泉, 高磊, 刘守仁, 王新华. X染色体一处新发现的SNP位点在脂尾(臀)、瘦尾绵羊群体中的多态检测及分析[J]. 西南农业学报, 2013, 26(5):2066-2070.doi:10.16213/j.cnki.scjas.2013.05.075.
	Gan S Q, Zhang W, Song T Z, Shen M, Liang Y W, Yang J Q, Gao L, Liu S R, Wang X H. Polymoriphism detection and analysis of novel SNP on X chromosome between fat-tailed and thin-tailed sheep breeds[J]. Southwest China Journal of Agricultural Sciences, 2013, 26(5):2066-2070.
[5]	Shi N, Zhang S, Guo Y D, Yu X H, Zhao W Z, Zhang M L, Guan Z H, Duan M. CircRNA_0050463 promotes Influenza A virus replication by sponging miR-33b-5p to regulate EEF1A1[J]. Veterinary Microbiology, 2021, 254:108995.doi:10.1016/j.vetmic.2021.108995.
[6]	Patop I L, Wüst S, Kadener S. Past,present,and future of circRNAs[J]. The EMBO Journal, 2019, 38(16):e100836.doi:10.15252/embj.2018100836.
[7]	Kim E, Kim Y K, Lee S V. Emerging functions of circular RNA in aging[J]. Trends in Genetics, 2021, 37(9):819-829.doi:10.1016/j.tig.2021.04.014. pmid: 34016449
[8]	董新星, 刘金桥, 李明丽, 严达伟, 王孝义, 陈强, 鲁绍雄. 撒坝猪与大白猪背脂差异circRNA筛选与调控网络分析[J]. 中国畜牧杂志, 2023, 59(5):116-123.doi:10.19556/j.0258-7033.20220711-05.
	Dong X X, Liu J Q, Li M L, Yan D W, Wang X Y, Chen Q, Lu S X. Screening of circRNA and analysis of regulatory network for back fat difference between Saba pig and large white pig[J]. Chinese Journal of Animal Science, 2023, 59(5):116-123.
[9]	李嫒, 张秀秀, 黄万龙, 解领丽, 苗向阳. 大白猪和莱芜猪肌内脂肪组织circRNAs的鉴定与分析[J]. 畜牧兽医学报, 2018, 49(7):1343-1353.doi:10.11843/j.issn.0366-6964.2018.07.003.
	Li A, Zhang X X, Huang W L, Xie L L, Miao X Y. Identification and analysis of circRNAs in intramuscular adipose tissues between large white and Laiwu pigs[J]. Chinese Journal of Animal and Veterinary Sciences, 2018, 49(7):1343-1353.
[10]	Huang M J, Shen Y F, Mao H G, Chen L X, Chen J C, Guo X L, Xu N Y. Circular RNA expression profiles in the porcine liver of two distinct phenotype pig breeds[J]. Asian-Australasian Journal of Animal Sciences, 2018, 31(6):812-819.doi:10.5713/ajas.17.0651. pmid: 29268579
[11]	曹海港, 葛静, 张小玉, 史新娥. 环状RNA及其在猪上的研究进展[J]. 畜牧与兽医, 2018, 50(12):129-132.
	Cao H G, Ge J, Zhang X Y, Shi X E. Circular RNA and progress in research on it in pigs[J]. Animal Husbandry & Veterinary Medicine, 2018, 50(12):129-132.
[12]	Liu T Y, Feng H, Yousuf S, Xie L L, Miao X Y. Functional analysis of differentially expressed circular RNAs in sheep subcutaneous fat[J]. BMC Genomics, 2023, 24(1):591.doi:10.1186/s12864-023-09401-6.
[13]	赵乐. 敖汉细毛羊肌内脂肪沉积相关circRNA的筛选与功能鉴定[D]. 青岛: 青岛农业大学, 2022. doi:10.27203/d.cnki.glync.2022.000021.
	Zhao L. Screening and functional identification of circRNA related to intramuscular fat deposition in Aohan fine wool sheep[D]. Qingdao: Qingdao Agricultural University, 2022.
[14]	Li B J, He Y, Wu W J, Tan X F, Wang Z, Irwin D M, Wang Z, Zhang S Y. Circular RNA profiling identifies novel circPPARA that promotes intramuscular fat deposition in pigs[J]. Journal of Agricultural and Food Chemistry, 2022, 70(13):4123-4137.doi:10.1021/acs.jafc.1c07358.
[15]	Wu J Y, Zhang S L, Yue B L, Zhang S H, Jiang E H, Chen H, Lan X Y. CircRNA profiling reveals CircPPARγ modulates adipogenic differentiation via sponging miR-92a-3p[J]. Journal of Agricultural and Food Chemistry, 2022, 70(22):6698-6708.doi:10.1021/acs.jafc.2c01815.
[16]	Jiang R, Li H, Yang J M, Shen X M, Song C C, Yang Z X, Wang X G, Huang Y Z, Lan X Y, Lei C Z, Chen H. CircRNA profiling reveals an abundant circFUT10 that promotes adipocyte proliferation and inhibits adipocyte differentiation via sponging let-7[J]. Molecular Therapy-Nucleic Acids, 2020, 20:491-501.doi:10.1016/j.omtn.2020.03.011. pmid: 32305019
[17]	Wang L D, Liang W S, Wang S S, Wang Z X, Bai H, Jiang Y, Bi Y L, Chen G H, Chang G B. Circular RNA expression profiling reveals that circ-PLXNA1 functions in duck adipocyte differentiation[J]. PLoS One, 2020, 15(7):e0236069.doi:10.1371/journal.pone.0236069.
[18]	Gao Y H, Wang S Z, Ma Y F, Lei Z X, Ma Y. Circular RNA regulation of fat deposition and muscle development in cattle[J]. Veterinary Medicine and Science, 2022, 8(5):2104-2113.doi:10.1002/vms3.857. pmid: 35689831
[19]	高鸿蒙, 张秀英, 张慧泽, 哈斯其木格, 周旭光, 魏健民, 于中正. 乌珠穆沁羊LPL基因第3外显子多态性与肉质性状的关联分析[J]. 农业生物技术学报, 2019, 27(3):449-456.doi:10.3969/j.issn.1674-7968.2019.03.008.
	Gao H M, Zhang X Y, Zhang H Z, Ha S, Zhou X G, Wei J M, Yu Z Z. Association analysis between polymorphism in the exon 3 of LPL gene and meat quality traits in ujimqin sheep(Ovis aries)[J]. Journal of Agricultural Biotechnology, 2019, 27(3):449-456.
[20]	王金泉, 王肖燕, 臧长江, 刘武军, 彭华刚, 王琦, 岳晓帆, 姚刚. 不同日龄阿勒泰大尾羊脂肪组织形态学及血脂指标的比较[J]. 中国畜牧兽医, 2014, 41(12):167-171.
	Wang J Q, Wang X Y, Zang C J, Liu W J, Peng H G, Wang Q, Yue X F, Yao G. Comparison of the serum lipids index and morphology of adipose tissue in different old Aletai sheep[J]. China Animal Husbandry & Veterinary Medicine, 2014, 41(12):167-171.
[21]	谢芳, 罗君谊, 陈婷, 习欠云, 张永亮, 孙加节. 非编码RNA调控猪肌间脂肪沉积的研究进展[J]. 中国畜牧兽医, 2023, 50(10):4133-4140.doi:10.16431/j.cnki.1671-7236.2023.10.027.
	Xie F, Luo J Y, Chen T, Xi Q Y, Zhang Y L, Sun J J. Research progress on non-coding RNA regulating intermuscular fat deposition in pig[J]. China Animal Husbandry & Veterinary Medicine, 2023, 50(10):4133-4140.
[22]	雷召雄, 魏大为, 汪书哲, 杨梦丽, 马云. 牛PLIN1基因CDS区扩增及时序表达[J]. 西北农业学报, 2020, 29(10):1472-1478.doi:10.7606/j.issn.1004-1389.2020.10.003.
	Lei Z X, Wei D W, Wang S Z, Yang M L, Ma Y. Coding region sequence and temporal expression of bovine PLIN1 gene[J]. Acta Agriculturae Boreali-occidentalis Sinica, 2020, 29(10):1472-1478.
[23]	Zhang Y F, Guo X, Pei J, Chu M, Ding X Z, Wu X Y, Liang C, Yan P. CircRNA expression profile during yak adipocyte differentiation and screen potential circRNAs for adipocyte differentiation[J]. Genes, 2020, 11(4):414.doi:10.3390/genes11040414.
[24]	Memczak S, Jens M, Elefsinioti A, Torti F, Krueger J, Rybak A, Maier L, Mackowiak S D, Gregersen L H, Munschauer M, Loewer A, Ziebold U, Landthaler M, Kocks C le Noble F, Rajewsky N. Circular RNAs are a large class of animal RNAs with regulatory potency[J]. Nature, 2013, 495(7441):333-338.doi:10.1038/nature11928.
[25]	Jeck W R, Sorrentino J A, Wang K, Slevin M K, Burd C E, Liu J Z, Marzluff W F, Sharpless N E. Circular RNAs are abundant,conserved,and associated with ALU repeats[J]. RNA, 2013, 19(2):141-157.doi:10.1261/rna.035667.112.
[26]	Salzman J, Chen R E, Olsen M N, Wang P L, Brown P O. Cell-type specific features of circular RNA expression[J]. PLoS Genetics, 2013, 9(9):e1003777.doi:10.1371/journal.pgen.1003777.
[27]	Shen Y D, Guo X W, Wang W M. Identification and characterization of circular RNAs in zebrafish[J]. FEBS Letters, 2017, 591(1):213-220.doi:10.1002/1873-3468.12500. pmid: 27878987
[28]	Lu T T, Cui L L, Zhou Y, Zhu C R, Fan D L, Gong H, Zhao Q, Zhou C C, Zhao Y, Lu D F, Luo J H, Wang Y C, Tian Q L, Feng Q, Huang T, Han B. Transcriptome-wide investigation of circular RNAs in rice[J]. RNA, 2015, 21(12):2076-2087.doi:10.1261/rna.052282.115. pmid: 26464523
[29]	Kos A, Dijkema R, Arnberg A C, van der Meide P H, Schellekens H. The Hepatitis delta(δ)virus possesses a circular RNA[J]. Nature, 1986, 323:558-560.doi:10.1038/323558a0.
[30]	Danan M, Schwartz S, Edelheit S, Sorek R. Transcriptome-wide discovery of circular RNAs in Archaea[J]. Nucleic Acids Research, 2012, 40(7):3131-3142.doi:10.1093/nar/gkr1009. pmid: 22140119
[31]	Guo R, Chen D F, Chen H Z, Fu Z M, Xiong C L, Hou C S, Zheng Y Z, Guo Y L, Wang H P, Du Y, Diao Q Y. Systematic investigation of circular RNAs in Ascosphaera apis,a fungal pathogen of honeybee larvae[J]. Gene, 2018, 678:17-22.doi:10.1016/j.gene.2018.07.076.
[32]	Zhu C Y, Cheng H P, Li N, Liu T G, Ma Y J. Isobaric tags for relative and absolute quantification-based proteomics reveals candidate proteins of fat deposition in Chinese indigenous sheep with morphologically different tails[J]. Frontiers in Genetics, 2021, 12:710449.doi:10.3389/fgene.2021.710449.

选择文件类型/文献管理软件名称

选择包含的内容