生物技术通报 ›› 2023, Vol. 39 ›› Issue (2): 274-282.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0826
李彦霞1,2(), 王晋鹏1,2, 冯芬1,2, 包斌武1,2, 董益闻1,2, 王兴平1,2, 罗仍卓么1,2()
收稿日期:
2022-07-04
出版日期:
2023-02-26
发布日期:
2023-03-07
作者简介:
李彦霞,女,硕士,研究方向:动物分子遗传育种;E-mail: 基金资助:
LI Yan-xia1,2(), WANG Jin-peng1,2, FENG Fen1,2, BAO Bin-wu1,2, DONG Yi-wen1,2, WANG Xing-ping1,2, LUORENG Zhuo-ma1,2()
Received:
2022-07-04
Published:
2023-02-26
Online:
2023-03-07
摘要:
旨为从基因水平上探究大肠杆菌(Escherichia coli)型乳房炎奶牛的产奶量和乳品质下降的原因。本实验利用脂多糖(LPS)诱导乳腺上皮细胞(bMECs)建立细胞炎症模型,并采用实时荧光定量PCR技术检测了奶牛产奶量与乳蛋白相关基因(CSN1S1、CSN2和LALBA)、乳脂调控相关基因(FABP3和LPL)以及炎症与产奶量相关基因MFGE8在奶牛乳腺组织和bMECs炎症反应中的表达量。结果表明,与对照组(0 h)相比,上述6个基因在E.coli型乳房炎奶牛的乳腺组织中表达量均极显著下调(P < 0.01)。此外,上述6个基因在LPS诱导bMECs产生炎症反应的3、6、12和24 h的bMECs中表达量也均极显著下调(P < 0.01)。在E.coli型奶牛乳房炎发生或bMECs产生炎症后,上述6个基因的表达水平均受到抑制,解释了E.coli型乳房炎导致产奶量减少、乳蛋白率和乳脂率下降的原因。
李彦霞, 王晋鹏, 冯芬, 包斌武, 董益闻, 王兴平, 罗仍卓么. 大肠杆菌型奶牛乳房炎对产奶性状相关基因表达的影响[J]. 生物技术通报, 2023, 39(2): 274-282.
LI Yan-xia, WANG Jin-peng, FENG Fen, BAO Bin-wu, DONG Yi-wen, WANG Xing-ping, LUORENG Zhuo-ma. Effects of Escherichia coli Dairy Cow Mastitis on the Expressions of Milk-producing Trait Related Genes[J]. Biotechnology Bulletin, 2023, 39(2): 274-282.
基因名称Gene name | GenBank登录号GenBank ID | 引物序列Primer sequence(5'-3') | 片段大小Product length/bp |
---|---|---|---|
IL-6[ | NM_173923.2 | F:CTGGGTTCAATCAGGCGAT R:CAGCAGGTCAGTGTTTGTGG | 205 |
IL-8[ | JN559767.1 | F:ACACATTCCACACCTTTCCAC R:ACCTTCTGCACCCACTTTTC | 149 |
TNF-α | NM_173966.3 | F:TCTGGGCAGGTCTACTTTG R:CCTGAGCCCATAATTCCCT | 139 |
CSN1S1 | NM_181029 | F:ATGTGCCCTCTGAGCGTTAC R:AGGCACCAGATGGATAGGC | 237 |
CSN2 | XM_015471671.2 | F:CTATGGCTCCTAAGCACAAAGA R: GCAGAGGCAGAGGAAGGTG | 117 |
LPL | NM_001075120 | F:GTTTATGAACTGGATGGCGG R:GAGAAAGGCGACTTGGAGC | 188 |
FABP3 | NM_174313 | F:TACTTACGAGAAACAGGCATGA R:CCTCAGAGCACCCTTTGG | 232 |
LALBA | NM_174378 | F:AAAGACGACCAGAACCCTCA R:GAAAGAGGACAGAAGCAGCAA | 223 |
MFGE8 | NM_176610 | F:CCCGCCCTCCTGATTGTA R:CCCACCGTGCCTAAGAAAA | 141 |
RPS18[ | NM_001033614.2 | F:GTGGTGTTGAGGAAAGCAGACA R:TGATCACACGTTCCACCTCATC | 79 |
GAPDH[ | NM_001034034.2 | F:GGCATCGTGGAGGGACTTATG R:GCCAGTGAGCTTCCCGTTGAG | 186 |
表1 qPCR引物信息
Table1 qPCR primer information
基因名称Gene name | GenBank登录号GenBank ID | 引物序列Primer sequence(5'-3') | 片段大小Product length/bp |
---|---|---|---|
IL-6[ | NM_173923.2 | F:CTGGGTTCAATCAGGCGAT R:CAGCAGGTCAGTGTTTGTGG | 205 |
IL-8[ | JN559767.1 | F:ACACATTCCACACCTTTCCAC R:ACCTTCTGCACCCACTTTTC | 149 |
TNF-α | NM_173966.3 | F:TCTGGGCAGGTCTACTTTG R:CCTGAGCCCATAATTCCCT | 139 |
CSN1S1 | NM_181029 | F:ATGTGCCCTCTGAGCGTTAC R:AGGCACCAGATGGATAGGC | 237 |
CSN2 | XM_015471671.2 | F:CTATGGCTCCTAAGCACAAAGA R: GCAGAGGCAGAGGAAGGTG | 117 |
LPL | NM_001075120 | F:GTTTATGAACTGGATGGCGG R:GAGAAAGGCGACTTGGAGC | 188 |
FABP3 | NM_174313 | F:TACTTACGAGAAACAGGCATGA R:CCTCAGAGCACCCTTTGG | 232 |
LALBA | NM_174378 | F:AAAGACGACCAGAACCCTCA R:GAAAGAGGACAGAAGCAGCAA | 223 |
MFGE8 | NM_176610 | F:CCCGCCCTCCTGATTGTA R:CCCACCGTGCCTAAGAAAA | 141 |
RPS18[ | NM_001033614.2 | F:GTGGTGTTGAGGAAAGCAGACA R:TGATCACACGTTCCACCTCATC | 79 |
GAPDH[ | NM_001034034.2 | F:GGCATCGTGGAGGGACTTATG R:GCCAGTGAGCTTCCCGTTGAG | 186 |
图1 CSN1S1、CSN2和LALBA基因在E.coli型乳房炎奶牛的乳腺组织中的表达量 ****表示P < 0.000 1;E.coli表示E.coli型乳房炎奶牛的乳腺组织;control表示健康奶牛的乳腺组织。下同
Fig. 1 Expressions of CSN1S1, CSN2 and LALBA gene in the cow mammary tissues of dairy cows with E.coli type mastitis **** refers to P < 0.000 1,E.coli means mammary tissue of cow with E.coli type mastitis; control means mammary tissue of healthy cow. The same below
图4 主要炎症因子基因在LPS诱导bMECs不同时间的表达量 A:体外培养的bMECs(200×);B:TNF-α的相对表达量;C:IL-6的相对表达量;D:IL-8的相对表达量。*P < 0.05;**P < 0.01;****P < 0.000 1,下同
Fig. 4 Expression of main inflammatory factor genes in LPS-induced bMECs at different times A: bMECs cultured in vitor(200×). B: The relative expression level of TNF-α. C: The relative expression of IL-6. D: The relative expression level of IL-8.*P < 0.5; **P < 0.01; and ****P < 0.000 1. The same below
图5 乳蛋白相关基因在LPS诱导bMECs不同时间的表达量 A:CSN1S1的相对表达量;B:CSN2的相对表达量;C:LALBA的相对表达量
Fig. 5 Expressions of milk protein-related genes in LPS-induced bMECs at different times A: Relative expression of CSN1S1. B: The relative expression of CSN2. C: The relative expression of LALBA
图6 乳脂调控相关基因在LPS诱导bMECs不同时间的表达量 A:FABP3的相对表达量;B:LPL的相对表达量
Fig. 6 Expressions of milk fat regulation related genes in LPS-induced bMECs at different times A: Relative expression level of FABP3; B: The relative expression level of LPL
[1] |
le Maréchal C, Thiéry R, Vautor E, et al. Mastitis impact on technological properties of milk and quality of milk products—a review[J]. Dairy Sci Technol, 2011, 91(3): 247-282.
doi: 10.1007/s13594-011-0009-6 URL |
[2] | 卢世杰, 吴红翔, 习学勇, 等. 奶牛乳房炎对主要牛奶成分的影响[J]. 中国奶牛, 2019(5): 27-32. |
Lu SJ, Wu HX, Xi XY, et al. Effect of cow mastitis on major milk components[J]. China Dairy Cattle, 2019(5): 27-32. | |
[3] |
Ju ZH, Jiang Q, Wang JP, et al. Genome-wide methylation and transcriptome of blood neutrophils reveal the roles of DNA methylation in affecting transcription of protein-coding genes and miRNAs in E. coli-infected mastitis cows[J]. BMC Genomics, 2020, 21(1): 102.
doi: 10.1186/s12864-020-6526-z URL |
[4] |
Blum SE, Heller ED, Leitner G. Long term effects of Escherichia coli mastitis[J]. Vet J, 2014, 201(1): 72-77.
doi: 10.1016/j.tvjl.2014.04.008 URL |
[5] |
Liu HY, Kai LX, Du HH, et al. LPS inhibits fatty acid absorption in enterocytes through TNF-α secreted by macrophages[J]. Cells, 2019, 8(12): 1626.
doi: 10.3390/cells8121626 URL |
[6] |
He XX, Liu WJ, Shi MY, et al. Docosahexaenoic acid attenuates LPS-stimulated inflammatory response by regulating the PPARγ/NF-κB pathways in primary bovine mammary epithelial cells[J]. Res Vet Sci, 2017, 112: 7-12.
doi: S0034-5288(16)30149-7 pmid: 28095338 |
[7] |
Luoreng ZM, Wang XP, Mei CG, et al. Expression profiling of peripheral blood miRNA using RNAseq technology in dairy cows with Escherichia coli-induced mastitis[J]. Sci Rep, 2018, 8(1): 12693.
doi: 10.1038/s41598-018-30518-2 URL |
[8] |
Luoreng ZM, Wang XP, Mei CG, et al. Comparison of microRNA profiles between bovine mammary glands infected with Staphylococcus aureus and Escherichia coli[J]. Int J Biol Sci, 2018, 14(1): 87-99.
doi: 10.7150/ijbs.22498 URL |
[9] |
Bionaz M, Loor JJ. ACSL1, AGPAT6, FABP3, LPIN1, and SLC27A6 are the most abundant isoforms in bovine mammary tissue and their expression is affected by stage of lactation[J]. J Nutr, 2008, 138(6): 1019-1024.
doi: 10.1093/jn/138.6.1019 URL |
[10] | Liu XL, Shen JL, Zong JX, et al. Beta-sitosterol promotes milk protein and fat syntheses-related genes in bovine mammary epithelial cells[J]. Animals(Basel), 2021, 11(11): 3238. |
[11] |
Song N, Chen YT, Luo J, et al. Negative regulation of αS1-casein(CSN1S1)improves β-casein content and reduces allergy potential in goat milk[J]. J Dairy Sci, 2020, 103(10): 9561-9572.
doi: 10.3168/jds.2020-18595 URL |
[12] |
Atabai K, Fernandez R, Huang XZ, et al. Mfge8 is critical for mammary gland remodeling during involution[J]. Mol Biol Cell, 2005, 16(12): 5528-5537.
pmid: 16195353 |
[13] |
Sabha BH, Alzahrani F, Almehdar HA, et al. Disorder in milk proteins: lactadherin multifunctionality and structure[J]. Curr Protein Pept Sci, 2018, 19(10): 983-997.
doi: 10.2174/1389203719666180608091849 URL |
[14] | 罗仍卓么, 王晋鹏, 焦鹏, 等. 奶牛乳腺炎模型的建立及炎症相关因子基因mRNA转录水平的分析[J]. 畜牧兽医学报, 2022, 53(8): 2763-2772. |
Luo R, Wang JP, Jiao P, et al. Construction of dairy cow mastitis model and analysis of mRNA transcription level of inflammation related cytokine genes[J]. Acta Vet Zootechnica Sin, 2022, 53(8): 2763-2772. | |
[15] |
Yang J, Hu QC, Wang JP, et al. RNA-seq reveals the role of miR-29c in regulating inflammation and oxidative stress of bovine mammary epithelial cells[J]. Front Vet Sci, 2022, 9: 865415.
doi: 10.3389/fvets.2022.865415 URL |
[16] |
Wang XP, Luoreng ZM, Zan LS, et al. Bovine miR-146a regulates inflammatory cytokines of bovine mammary epithelial cells via targeting the TRAF6 gene[J]. J Dairy Sci, 2017, 100(9): 7648-7658.
doi: 10.3168/jds.2017-12630 URL |
[17] | 焦鹏, 王兴平, 汪书哲, 等. 奶牛乳腺炎差异表达lncRNA BCL2对炎症及凋亡相关mRNA表达的影响[J]. 畜牧兽医学报, 2022, 53(7): 2160-2171. |
Jiao P, Wang XP, Wang SZ, et al. Effect of differentially expressed lncRNA BCL2 in dairy cow with mastitis on the expression of inflammation and apoptosis related mRNA[J]. Acta Vet Zootechnica Sin, 2022, 53(7): 2160-2171. | |
[18] | 李宇航, 王兴平, 杨箭, 等. miR-665在奶牛乳腺上皮细胞炎症中的表达及功能分析[J]. 生物技术通报, 2022, 38(5): 159-168. |
Li YH, Wang XP, Yang J, et al. Expression and functional analysis of miR-665 in bovine mammary epithelial cell inflammation[J]. Biotechnol Bull, 2022, 38(5): 159-168. | |
[19] |
Liu MJ, Zhang C, Xu XL, et al. Ferulic acid inhibits LPS-induced apoptosis in bovine mammary epithelial cells by regulating the NF-κB and Nrf2 signalling pathways to restore mitochondrial dynamics and ROS generation[J]. Vet Res, 2021, 52(1): 104.
doi: 10.1186/s13567-021-00973-3 pmid: 34256834 |
[20] |
Sharun K, Dhama K, Tiwari R, et al. Advances in therapeutic and managemental approaches of bovine mastitis: a comprehensive review[J]. Vet Q, 2021, 41(1): 107-136.
doi: 10.1080/01652176.2021.1882713 pmid: 33509059 |
[21] | 李莲, 唐娟, 吴洁, 等. 基于iTRAQ技术的脂多糖刺激下奶牛乳腺上皮细胞的蛋白组学分析[J]. 畜牧兽医学报, 2016, 47(9): 1853-1860. |
Li L, Tang J, Wu J, et al. Analysis of expression protein profiles of bovine mammary epithelial cell based on isobaric tag for relative and absolute quantification proteomics and bioinformatics technique[J]. Chin J Animal Vet Sci, 2016, 47(9): 1853-1860. | |
[22] |
Chen JB, Wu YJ, Sun YW, et al. Bacterial endotoxin decreased histone H3 acetylation of bovine mammary epithelial cells and the adverse effect was suppressed by sodium butyrate[J]. BMC Vet Res, 2019, 15(1): 267.
doi: 10.1186/s12917-019-2007-5 pmid: 31357995 |
[23] |
Visker MHPW, Dibbits BW, Kinders SM, et al. Association of bovine β-casein protein variant I with milk production and milk protein composition[J]. Anim Genet, 2011, 42(2): 212-218.
doi: 10.1111/j.1365-2052.2010.02106.x pmid: 24725229 |
[24] |
Dixit SP, Singh S, Ganguly I, et al. Genome-wide runs of homozygosity revealed selection signatures in Bos Indicus[J]. Front Genet, 2020, 11: 92.
doi: 10.3389/fgene.2020.00092 pmid: 32153647 |
[25] |
Wu YJ, Chen JB, Sun YW, et al. PGN and LTA from Staphylococcus aureus induced inflammation and decreased lactation through regulating DNA methylation and histone H3 acetylation in bovine mammary epithelial cells[J]. Toxins, 2020, 12(4): 238.
doi: 10.3390/toxins12040238 URL |
[26] | 赵笑, 白沙沙, 孔凡华, 等. 乳蛋白功能特性及其分析检测技术研究进展[J]. 中国乳品工业, 2022, 50(1): 37-42. |
Zhao X, Bai SS, Kong FH, et al. Research progress on the functional properties of milk protein and its analytical detection technology[J]. China Dairy Ind, 2022, 50(1): 37-42. | |
[27] |
Neville MC. Introduction: alpha-lactalbumin, a multifunctional protein that specifies lactose synthesis in the Golgi[J]. J Mammary Gland Biol Neoplasia, 2009, 14(3): 211-212.
doi: 10.1007/s10911-009-9149-1 URL |
[28] | 单曦, 吉丽, 张永云, 等. 槟榔江水牛LALBA基因克隆及序列特征分析[J]. 云南农业大学学报: 自然科学, 2017, 32(5): 830-837. |
Shan X, Ji L, Zhang YY, et al. Cloning, sequence characterization of Binglangjiang buffalo gene LALBA[J]. J Yunnan Agric Univ Nat Sci, 2017, 32(5): 830-837. | |
[29] | Spitzer AJ, Tian Q, Choudhary RK, et al. Bacterial endotoxin induces oxidative stress and reduces milk protein expression and hypoxia in the mouse mammary gland[J]. Oxid Med Cell Longev, 2020, 2020: 3894309. |
[30] |
Li AN, Wu LJ, Wang XY, et al. Tissue expression analysis, cloning and characterization of the 5'-regulatory region of the bovine FABP3 gene[J]. Mol Biol Rep, 2016, 43(9): 991-998.
doi: 10.1007/s11033-016-4026-7 URL |
[31] |
Zhao WS, Hu SL, Yu K, et al. Lipoprotein lipase, tissue expression and effects on genes related to fatty acid synthesis in goat mammary epithelial cells[J]. Int J Mol Sci, 2014, 15(12): 22757-22771.
doi: 10.3390/ijms151222757 URL |
[32] |
Liang MY, Hou XM, Qu B, et al. Functional analysis of FABP3 in the milk fat synthesis signaling pathway of dairy cow mammary epithelial cells[J]. In Vitro Cell Dev Biol Anim, 2014, 50(9): 865-873.
doi: 10.1007/s11626-014-9780-z URL |
[33] |
Yang YW, Pan QQ, Sun BX, et al. miR-29b targets LPL and TDG genes and regulates apoptosis and triglyceride production in MECs[J]. DNA Cell Biol, 2016, 35(12): 758-765.
doi: 10.1089/dna.2016.3443 URL |
[34] |
Zhong WQ, Shen JL, Liao XD, et al. Camellia(Camellia oleifera Abel.)seed oil promotes milk fat and protein synthesis-related gene expression in bovine mammary epithelial cells[J]. Food Sci Nutr, 2019, 8(1): 419-427.
doi: 10.1002/fsn3.1326 URL |
[35] |
Fitz-Gerald CH, Deeth HC, Kitchen BJ. The relationship between the levels of free fatty acids, lipoprotein lipase, carboxylesterase, N-acetyl-beta-D-glucosaminidase, somatic cell count and other mastitis indices in bovine milk[J]. J Dairy Res, 1981, 48(2): 253-265.
pmid: 7298962 |
[36] |
Hanayama R, Nagata S. Impaired involution of mammary glands in the absence of milk fat globule EGF factor 8[J]. Proc Natl Acad Sci USA, 2005, 102(46): 16886-16891.
pmid: 16275924 |
[37] |
Nakatani H, Aoki N, Nakagawa Y, et al. Weaning-induced expression of a milk-fat globule protein, MFG-E8, in mouse mammary glands, as demonstrated by the analyses of its mRNA, protein and phosphatidylserine-binding activity[J]. Biochem J, 2006, 395(1): 21-30.
pmid: 16401186 |
[1] | 杨志晓, 侯骞, 刘国权, 卢志刚, 曹毅, 芶剑渝, 王轶, 林英超. 不同抗性烟草品系Rubisco及其活化酶对赤星病胁迫的响应[J]. 生物技术通报, 2023, 39(9): 202-212. |
[2] | 李帜奇, 袁月, 苗荣庆, 庞秋颖, 张爱琴. 盐胁迫盐芥和拟南芥褪黑素含量及合成相关基因表达模式分析[J]. 生物技术通报, 2023, 39(5): 142-151. |
[3] | 刘奎, 李兴芬, 杨沛欣, 仲昭晨, 曹一博, 张凌云. 青杄转录共激活因子PwMBF1c的功能研究与验证[J]. 生物技术通报, 2023, 39(5): 205-216. |
[4] | 赖瑞联, 冯新, 高敏霞, 路喻丹, 刘晓驰, 吴如健, 陈义挺. 猕猴桃过氧化氢酶基因家族全基因组鉴定与表达分析[J]. 生物技术通报, 2023, 39(4): 136-147. |
[5] | 郭三保, 宋美玲, 李灵心, 尧子钊, 桂明明, 黄胜和. 斑地锦查尔酮合酶基因及启动子的克隆与分析[J]. 生物技术通报, 2023, 39(4): 148-156. |
[6] | 陈强, 邹明康, 宋家敏, 张冲, 吴隆坤. 甜瓜LBD基因家族的鉴定和果实发育进程中的表达分析[J]. 生物技术通报, 2023, 39(3): 176-183. |
[7] | 姚晓文, 梁晓, 陈青, 伍春玲, 刘迎, 刘小强, 税军, 乔阳, 毛奕茗, 陈银华, 张银东. 二斑叶螨抗性木薯木质素合成途径基因表达特性研究[J]. 生物技术通报, 2023, 39(2): 161-171. |
[8] | 冯策婷, 江律, 刘鑫颖, 罗乐, 潘会堂, 张启翔, 于超. 单叶蔷薇NAC基因家族鉴定及干旱胁迫响应分析[J]. 生物技术通报, 2023, 39(11): 283-296. |
[9] | 吴柏增, 何琪, 姚方杰, 赵梦然. 糙皮侧耳乳酸脱氢酶鉴定及其菌丝高温胁迫下表达特征分析[J]. 生物技术通报, 2023, 39(11): 350-359. |
[10] | 姜南, 石杨, 赵志慧, 李斌, 赵熠辉, 杨俊彪, 闫家铭, 靳雨璠, 陈稷, 黄进. 镉胁迫下水稻OsPT1的表达及功能分析[J]. 生物技术通报, 2023, 39(1): 166-174. |
[11] | 段敏杰, 李怡斐, 杨小苗, 王春萍, 黄启中, 黄任中, 张世才. 辣椒锌指蛋白DnaJ-Like基因家族鉴定及对高温胁迫的表达响应[J]. 生物技术通报, 2023, 39(1): 187-198. |
[12] | 袁星, 郭彩华, 刘金明, 亢超, 全绍文, 牛建新. 核桃CONSTANS-Like基因家族全基因组鉴定及表达分析[J]. 生物技术通报, 2022, 38(9): 167-179. |
[13] | 郭宾会, 宋丽. 大豆孢囊线虫侵染对乙烯合成及信号传导基因表达调控的研究[J]. 生物技术通报, 2022, 38(8): 150-158. |
[14] | 张淼, 杨露露, 贾岩龙, 王天云. DNA甲基化和组蛋白甲基化修饰的表观遗传调控作用研究进展[J]. 生物技术通报, 2022, 38(7): 23-30. |
[15] | 李一涵, 于浪柳, 李春燕, 张蒙蒙, 张晓勤, 方云霞, 薛大伟. 大麦NRAMP全基因组鉴定及重金属胁迫下基因表达分析[J]. 生物技术通报, 2022, 38(6): 103-111. |
阅读次数 | ||||||
全文 |
|
|||||
摘要 |
|
|||||