Prediction and Experimental Verification of a Hierarchical Transcription Factor Regulatory Network of Porcine Myoglobin (Mb)

Abstract

Myoglobin is a key chemical component that determines meat's color and affects consumers' purchase intentions. In this work, we firstly identified the promoter sequence of the Mb gene from the primary assembly of high-throughput genome sequencing in pigs, and predicted its potential transcription factors by LASAGNA. Through the data mining of the mRNA expression profile of longissimus dorsi muscle of different pig breeds, we constructed a hierarchical interplay network of Mb-TFs (Myoglobin-Transcription Factors), consisting of 16 adaptive transcription factors and 23 secondary transcription factors. The verification of gene expression in longissimus dorsi muscle showed that the Mb mRNA and encoded protein were significantly (p < 0.05) more abundant in Bama pigs than Yorkshire pigs. The qRT-PCR (Real-Time Quantitative Reverse Transcription PCR) validation on genes of the Mb-TFs network showed that FOS, STAT3, STAT1, NEFL21, NFE2L2 and MAFB were significant positive regulatory core transcription factors of Mb-TFs network in Bama pigs, whereas ATF3 was the secondary transcription factor most responsible for the activation of the above transcription factors. Our study provides a new strategy to unravel the mechanism of pork color formation, based on public transcriptome and genome data analysis.

Keywords: Bama pigs; Yorkshire pigs; meat color; myoglobin; transcription factor.

Figure 1

The localization of Mb gene on the porcine reference genome. (A) genomic structure information of Mb transcripts on the primary assembly of Wuzhishan mini-pig; (B) the localization of Mb gene on chromosome 5 of the porcine reference genome.

Figure 2

Prediction and enrichment of transcription factors of porcine Mb gene. (A) multiple sequence alignment of promoters (2 kb) of Mb gene in 8 representative mammals; (B) heatmap of 62 TFs expression in 9 tissues of pig with binding sites integrated.

Figure 3

Prediction of adaptive transcription factors of porcine Mb gene and construction of its interaction network. (A) transcription factor interaction network of the porcine Mb gene. The network is constructed using the interaction data from the STRING database via the Cytoscape software. Red, myoglobin; green, core transcription factor; orange, secondary transcription factor. The size of the node (protein) indicates node connection degree (interaction relationships); (B) pathway enrichment of transcription factors of the porcine Mb gene.

Figure 4

Detection of meat color and Mb gene expression. (A) meat color detection of Yorkshire and Bama as measured by the Germany Matthaus meat color tester Opto-Star; (B) Mb mRNA expression of Yorkshire and Bama as measured by RT-qPCR; (C) Mb protein expression of Yorkshire and Bama as measured by Western blot (Original Western Blot Figures are in Figure S1). ** p < 0.05, **** p < 0.01.

Figure 5

qRT-PCR validation of transcription factors on the porcine Mb gene regulatory network of the Bama breed. (A) core transcription factor expression of the Bama breed in the Mb-TFs network; (B) secondary transcription factor expression of the Bama breed in the Mb-TFs network; (C) overview of transcription factor expression foldchange and binding sites number of the Bama breed in the Mb-TFs network. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: no significance.

Figure 5

qRT-PCR validation of transcription factors on the porcine Mb gene regulatory network of the Bama breed. (A) core transcription factor expression of the Bama breed in the Mb-TFs network; (B) secondary transcription factor expression of the Bama breed in the Mb-TFs network; (C) overview of transcription factor expression foldchange and binding sites number of the Bama breed in the Mb-TFs network. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: no significance.