过表达和干扰PRKD1基因对牛成骨细胞分化的影响

doi:10.11843/j.issn.0366-6964.2025.07.014

Abstract

Abstract:

The aim of this study was to investigate the regulatory effect of protein kinase D1 (PRKD1) gene, an important candidate gene for chest depth trait in beef carcass, on the osteogenic differentiation of bovine osteoblasts. In this study, osteoblasts derived from pregnancy Huaxi bovine fetal ribs with 3 months old were used as in vitro models. According to PRKD1 gene sequence, PRKD1 overexpressed plasmid (pPRKD1) and PRKD1 siRNA (siPRKD1) were constructed and synthesized, respectively, and transfected into bovine osteoblasts. On the 4 days of differentiation, RT-qPCR and Western blot assay were used to detect the expression levels of RUNX2, SP7, SPP1 and COL I genes. The activity of ALP was detected on the 7 days of differentiation. On the 21 days of differentiation, the osteogenic ability of osteoblasts was detected by Alizarin Red calcium nodule staining. All the above-mentioned experiments were conducted with 3 biological replicates each, and within each group, 3 intra-group replicates were set up. The results showed that after overexpression of PRKD1, mRNA and protein expressions of SP7 and COL I genes were significantly increased(P < 0.01), while RUNX2 and SPP1 genes were not significantly changed, PRKD1 overexpression also significantly increased the ALP activity and mineralized nodules of osteoblasts, while interference of PRKD1 showed the opposite effect. In conclusion, PRKD1 gene significantly affects the differentiation and osteogenic function of bovine rib osteoblasts, and the results provide theoretical basis for further analysis of the regulation mechanism of PRKD1 gene on bovine carcass chest depth traits.

Key words: bovine, PRKD1, osteoblasts, osteogenic differentiation

CLC Number:

S823.2

ZHAO Tong, WANG Yahui, WU Tianyi, GAO Chen, GAO Xiaoxiao, ZHANG Lupei, GAO Huijiang, LI Junya. Effects of Overexpression and Interference of PRKD1 Gene on Osteogenic Differentiation of Bovine Osteoblasts[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(7): 3188-3198.

Figures/Tables 8

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References 37

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siRNA名称Name	序列(5′→3′) Sequence
siPRKD1-1	CCGAAGAAACAGAAATGAA
siPRKD1-2	GCACATCAGCACAGTTTAT
siPRKD1-3	GCTGAATTACCACAAGAGA

基因Gene	上游引物(5′→3′) Forward primer sequence	下游引物(5′→3′) Reverse primer sequence
PRKD1	AGACTTTCAGATTCGCCCCC	TCAGCCCACATCCTTCACAT
RUNX2	CGGAGTGGAAGAGGCAAGAG	TGGATGGACGGAGGAGTCAT
COL I	AGGCCAAGAAGAAGACATCCC	GGGACTTTGGCGTTAGGACA
SPP1	GCTAAAGCCTGACCCATCTCA	GGACTTACTTGGGAGGGTATTTTG
SP7	TGGCACACGGGCGAGAGG	TGGAGCAGAGTAGGCAGGTGAAC
GAPDH	AGGTCGGAGTGAACGGATTC	CCAGCATCACCCCACTTGAT

细胞Cell	OCN水平/(ng·mL^-1) OCN level	ALP水平/(μmol·mL^-1) ALP level
成纤维细胞Fibroblasts	7.736±0.041 6	0.506±0.061 5
成骨细胞Osteoblasts	10.328±0.082 6^**	2.378±0.073 1^**

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Effects of Overexpression and Interference of PRKD1 Gene on Osteogenic Differentiation of Bovine Osteoblasts

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