Screening of target genes related to milk fat in dairy cows regulated by alfalfa miR168b

doi:10.11686/cyxb2022449

Abstract

Abstract:

The alfalfa （Medicago sativa） miRNAs expressed significantly differently in the blood and milk of high-fat dairy cows and low-fat dairy cows and their potential target genes in dairy cows were screened out in order to lay a foundation for further exploring the regulation of milk fat level by alfalfa miRNAs at gene level. Firstly， the level of dairy herd improvement （DHI） was measured in milk of Holstein cows which had produced 4 fetuses and had the same dietary level. Three cows were selected from high-and low-milk-fat cows as duplicates and RT-qPCR was used to quantify alfalfa miRNAs in milk and blood of the dairy cows. After screening out differentially expressed miRNAs， the target genes were predicted and analyzed， and the target genes related to milk fat metabolism were screened according to the binding sites and quantitative results by miRNA-mRNA. The results were as follows： 1） Three high-fat dairy cows and three low-fat dairy cows were identified by their DHI score. The fat content in the milk of high-fat dairy cows was >4.2%， and that of low-fat dairy cows was <3.5%. 2） Alfalfa sourced novel-miR54， miR156f， miR166a， miR168b and miR168c-3p were detected in cow blood and milk. The expression level of miR168b in the blood of high-fat milk cows was extremely significantly lower than that in low-fat milk cows （P<0.01）， and similarly the expression level of miR168b in the milk of high-fat milk cows was significantly lower than that in low-fat milk cows （P<0.05）. 3） The high expression of mtr-miR168b in mammary epithelial cells inhibited the expression of adipogenic marker genes PPARγ， SCD1， CEBP/β and SREBP1. 4） GO and KEGG databases indicated 1834 and 296 target genes， respectively， predicted by mtr-miR168b. The predicted target genes were closely related to N-glycan biosynthesis （3.72%）， the cGMP-PKG signaling pathway （2.37%） and vascular smooth muscle contraction （2.37%） and the glycerophosphate metabolism pathway （1.69%） was also significantly enriched. 5） CPT1A and STARD7， two genes closely related to lipid metabolism， were also screened. A targeting relationship between miR-168b and CPT1A was confirmed by dual-luciferase report. In conclusion， mtr-miR168b can regulate milk fat production by inhibiting the expression of adipogenic marker genes in mammary epithelial cells， and this study identifies target genes for further verification of alfalfa miRNA regulation of milk fat percentage in dairy cows.

Key words: Holstein cow, cross-kingdom regulation, mtr-miR168b, milk fat, target gene

Jing-ying JIA, Bao-bao LIU, Yun MA, Hong-juan DUAN, Xiao-yan CAI. Screening of target genes related to milk fat in dairy cows regulated by alfalfa miR168b[J]. Acta Prataculturae Sinica, 2023, 32(10): 173-186.

Figures/Tables 15

References 35

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牛号 ID	产犊日期 Calving date （year/month/day）	胎次 Parity	产犊间隔 Calving interval （d）	泌乳天数 Lactation days （d）
0001	2021/7/19	4	371	120
0002	2021/7/12	4	340	127
0003	2021/5/13	4	364	187
0004	2021/7/12	4	426	127
0005	2021/5/15	4	381	185
0006	2021/7/31	4	364	108

牛号 ID	产犊日期 Calving date （year/month/day）	胎次 Parity	产犊间隔 Calving interval （d）	泌乳天数 Lactation days （d）
0001	2021/7/19	4	371	120
0002	2021/7/12	4	340	127
0003	2021/5/13	4	364	187
0004	2021/7/12	4	426	127
0005	2021/5/15	4	381	185
0006	2021/7/31	4	364	108

项目 Item	含量 Content	营养水平 Nutritient level	含量 Content
苜蓿 Alfalfa （%）	6.00	干物质采食量 Dry matter feed intake（kg·d^-1）	24.4
玉米青贮 Corn silage （%）	50.00	粗蛋白 Crude protein （CP， %）	17.2
全棉籽 Cottonseed （%）	1.00	中性洗涤纤维 Neutral detergent fiber （NDF， %）	27.5
甜菜颗粒 Beet granules （%）	1.60	粗脂肪 Fat （%）	3.9
湿啤酒糟 Wet beer lees （%）	10.00	酸性洗涤纤维 Acid detergent fiber （ADF， %）	21.7
压片玉米 Tablet corn （%）	4.00	牛奶净能Milk net energy production （NEL， MJ·kg^-1）	7.78
预混料 Premix （%）	26.96	淀粉 Starch （%）	27.5
水 H₂O （%）	1.20	钙 Ca （%）	0.53
合计 Total （%）	100.00	磷 P （%）	0.36

项目 Item	含量 Content	营养水平 Nutritient level	含量 Content
苜蓿 Alfalfa （%）	6.00	干物质采食量 Dry matter feed intake（kg·d^-1）	24.4
玉米青贮 Corn silage （%）	50.00	粗蛋白 Crude protein （CP， %）	17.2
全棉籽 Cottonseed （%）	1.00	中性洗涤纤维 Neutral detergent fiber （NDF， %）	27.5
甜菜颗粒 Beet granules （%）	1.60	粗脂肪 Fat （%）	3.9
湿啤酒糟 Wet beer lees （%）	10.00	酸性洗涤纤维 Acid detergent fiber （ADF， %）	21.7
压片玉米 Tablet corn （%）	4.00	牛奶净能Milk net energy production （NEL， MJ·kg^-1）	7.78
预混料 Premix （%）	26.96	淀粉 Starch （%）	27.5
水 H₂O （%）	1.20	钙 Ca （%）	0.53
合计 Total （%）	100.00	磷 P （%）	0.36

miRNAs名称miRNAs name	引物序列Primer sequence （5'-3'）
U6	F：GCTTCGGCAGCACATATACTAAAAT
U6	R：CGCTTCACGAATTTGCGTGTCAT
mtr-miR168b	F：CATGTGTCGCTTGGTGCAG
	R：AGTGCAGGGTCCGAGGTATT
	RT：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTTCCCGAC
mtr-miR166a	F： CACAGTTCGGACCAGGCTT
	R： AGTGCAGGGTCCGAGGTATT
	RT： GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGGGAATG
mtr-miR168c-3p	F： CATAGACCCGCCTTGCATC
	R： AGTGCAGGGTCCGAGGTATT
	RT： GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACATTCAGTT
mtr-miR156f	F： CCGTTGACAGAAGATAGAGAGCAC
	R： ATCCAGTGCAGGGTCCGAGG
	RT： GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGTGCTC
mtr-novel-miR54	F： CCAAGTCCTTGTGTTGCATCTC
	R： ATCCAGTGCAGGGTCCGAGG
	RT： GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGAGATG
bta-miR-16a	F： GCCCGTAGCAGCACGTAAAT
	R： TGTCGTGGAGTCGGCAAT
	RT： CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCACCAA