QPCTL Affects the Daily Weight Gain of the F2 Population and Regulates Myogenic Cell Proliferation and Differentiation in Chickens

Abstract

Molecular breeding can accelerate the process of animal breeding and improve the breeding efficiency. To date, many Indel molecular markers have been identified in livestock and poultry, but how Indels affect economic traits is not well understood. For molecular breeding, it is crucial to reveal the mechanism of action of Indels and to provide more accurate information. The purpose of this study was to investigate how the 52/224-bp multiallelic Indels of the chicken QPCTL promoter area affect the daily weight gain of chickens and the potential regulatory mechanism of the QPCTL gene. The analysis was conducted by association analysis, qPCR, dual-fluorescence assay and Western blotting. The results showed that Indels in the QPCTL promoter region were significantly associated with the daily weight gain in chickens and that QPCTL expression showed a decreasing trend in embryonic breast muscle tissues. Furthermore, QPCTL expression was significantly higher in breast muscle tissues of the AC genotype than in those of the AB and BB genotypes. Based on the transcriptional activity results, the pGL3-C vector produced more luciferase activity than pGL3-A and pGL3-B. In addition, overexpression of QPCTL promoted chicken primary myoblast (CPM) proliferation and inhibited differentiation. The results of this study suggest that Indels in the promoter region of the QPCTL gene may regulate the proliferation and differentiation of CPMs by affecting the expression of QPCTL, which ultimately affects the growth rate of chickens. These Indels have important value for the molecular breeding of chickens, and QPCTL can be used as a candidate gene to regulate and improve chicken growth and development.

Keywords: Indel; QPCTL; cell proliferation; daily weight gain; primary myoblast.

Figure 1

Bioinformatics prediction of QPCTL. (a) Phylogenetic tree of chicken QPCTL. Gallus gallus (chicken), Colinus virginianus (northern bobwhite), Strigops habroptila (kakapo), Bos taurus (cattle), Capra hircus (goat), Sus scrofa (pig), Homo sapiens (human), Papio anubis (olive baboon), Mus musculus (house mouse), Rattus rattus (black rat), and Xenopus tropicalis (tropical clawed frog). (b) Interacting proteins of chicken QPCTL. The purple line represents the interaction that has been verified by experiments, the black line represents the co-expression relationship, the yellow line represents text mining, and the blue line represents the gene neighborhood.

Figure 2

Relative expression levels of the QPCTL gene. (a) Relative expression patterns of QPCTL in different tissues and at different weeks of age in Gushi chickens (n = 12). (b) Expression of the QPCTL gene in breast muscle at different embryonic stages in Xinghua chickens (n = 18). * p < 0.05; ** p < 0.01.

Figure 3

Effects of different Indels on QPCTL. (a) Expression of the QPCTL gene in breast muscle, leg muscle and abdominal fat of Gushi chickens with different genotypes (n = 12). We did not collect sufficient tissues from CC and BC genotype individuals to compare the expression of all genotypes. (b) Luciferase activity in DF-1 cells transfected with recombinant plasmids. * p < 0.05; ** p < 0.01.

Figure 4

Predicted binding sites of transcription factors in the A, B and C alleles; the A allele (insertions of 52 bp and 224 bp), the B allele (deletion of 52 bp and insertion of 224 bp) and the C allele (insertion of 52 bp). The −362 to −89 (ATG as +1) region existed in three Indels: 112 bp, 96 bp, and 16 bp. In our previous study, we found that these three Indels were simultaneously inserted or deleted in 1806 chickens by genotyping; thus, we regard them as a 224 bp Indel [8].

Figure 5

Trend analysis of QPCTL and MYOD gene expression. (a) Expression of QPCTL and MYOD genes in breast muscle of Gushi chickens fed a high-protein diet (n = 18). (b) Correlation analysis of QPCTL and MYOD gene expression in chickens fed a high-protein diet. (c) Expression of QPCTL and MYOD genes in breast muscle of Gushi chickens fed a high-fat diet (n = 10). (d) Correlation analysis of QPCTL and MYOD gene expression in chickens fed a high-fat diet. * p < 0.05; ** p < 0.01.

Figure 6

QPCTL promotes the proliferation of CPMs and inhibits their differentiation. (a) Expression of cell cycle-related genes after the overexpression of QPCTL. (b,c) Cell cycle analysis of QPCTL overexpression in CPMs. Count represents the number of cells counted; FL2-A is the propidium iodide fluorescence area signal, representing the DNA content of cells. The result of (c) comes from (b). (d) Expression of myoblast differentiation-related genes after the overexpression of QPCTL. (e,f) Protein level of MYOD in differentiated myoblasts transfected with the QPCTL overexpression vector. * p < 0.05; ** p < 0.01.