大肠杆菌型奶牛乳房炎对产奶性状相关基因表达的影响

doi:10.13560/j.cnki.biotech.bull.1985.2022-0826

摘要/Abstract

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。

关键词: Escherichia coli, 乳房炎, 产奶性状, 乳腺上皮细胞, 基因表达

Abstract:

This work aims to explore the reasons of milk yield and milk quality declining from dairy cows with Escherichia coli mastitis at the genetic level. This experiment established a cell inflammation model by lipopolysaccharide(LPS)-induced bovine mammary epithelial cells（bMECs）. The expressions of milk yield and milk protein-related genes（CSN1S1, CSN2 and LALBA）, milk fat regulation-related genes（FABP3 and LPL）and inflammation and milk yield-related gene MFGE8 were detected by real-time quantitative PCR（qPCR）. The results showed that the expressions of the above 6 genes were significantly down-regulated in the mammary tissues of dairy cows with E.coli mastitis（P < 0.01）compared with the control group（0 h）. In addition, the expressions of the six genes were also significantly down-regulated in LPS-induced bMECs at 3, 6, 12 and 24 h（P < 0.01）. This indicated that the expressions of the six genes were inhibited after E. coli mastitis or bMECs inflammation occurred, which explained the reasons for E.coli mastitis reducing milk yield, milk protein percentage and milk fat percentage.

Key words: Escherichia coli, mastitis, milk-producing trait, mammary epithelial cells, gene expression

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.

图/表 8

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^[17]	NM_173923.2	F：CTGGGTTCAATCAGGCGAT R：CAGCAGGTCAGTGTTTGTGG	205
IL-8^[18]	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^[18]	NM_001033614.2	F：GTGGTGTTGAGGAAAGCAGACA R：TGATCACACGTTCCACCTCATC	79
GAPDH^[18]	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2 FABP3和LPL基因在E.coli型乳房炎奶牛的乳腺组织中的表达量

Fig. 2 Expressions of FABP3 and LPL gene in the mam-mary tissues of dairy cows with E.coli mastitis

图3 MFGE8基因在E.coli型乳房炎奶牛的乳腺组织中的表达量

Fig. 3 Expression of MFGE8 gene in mammary tissue of dairy cows with E.coli mastitis

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

图7 MFGE8基因在LPS诱导bMECs不同时间的表达量

Fig. 7 Expressions of MFGE8 gene in LPS-induced bMECs at different times

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