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The SNPs in myoD gene from normal muscle developing individuals have no effect on muscle mass.
Ding S
,
Nie Y
,
Zhang X
,
Liu X
,
Yuan R
,
Chen K
,
Zhu Q
,
Cai S
,
Fang Y
,
Chen Y
,
Mo D
.
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BACKGROUND: Myogenic Differentiation 1 (MyoD) is a crucial master switch in regulating muscle-specific gene transcription. Forced expression of myoD is equipped to induce several cell lineages into myoblast, which then differentiate and fuse into myotube. Pig is one of the most significant livestock supplying meat, and has been classified into lean, fat and miniature pig breeds. However, the mechanisms underlying muscle mass variation among different pig breeds have remained unclear. Considering the important effect of MyoD on muscle development, it remains to be investigated whether the difference in muscle mass is caused by its single nucleotide polymorphisms (SNPs) which are the major differences among pig breeds at DNA level.
RESULTS: In this study, we identified the locations of porcine myoD regulatory regions including proximal regulatory region (PRR), distal regulatory region (DRR), and core enhancer (CE) region. There are 8 SNPs in the regulatory regions and 6 SNPs in gene body region, which were identified from lean, fat and miniature pig populations. However, these SNPs have no effects on its temporal expression and transcriptional activity which might lead to the distinction in postnatal muscle mass. In addition, overexpression of myoD clones across from amphibious to mammals including xenopus tropicalis, chicken, mouse and pig whose gene identities vary from 68 to 84%, could promote myogenesis in NIH3T3 fibroblasts cells.
CONCLUSIONS: These results proved that myoD nucleotide variations from different pig populations have no effect on muscle mass, suggesting that the function of myoD is highly conserved not only among different pig breeds, but also across different species. Thus, it would be futile to discover SNPs affecting muscle mass in pig populations with normal muscle development.
2018YFD0501200 National Key R&D Program of China, 31772565 National Natural Science Foundation of China, 2014YT02H042 Sailing Plan of Guangdong Province, CARS-36 National Swine Industry Technology System
Fig. 1. Sequence and location determination of myoD in pigs. (a-c) Homology of CE regions (a), DRR (b) and PRR (c) of humans, mice and pigs myoD were obtained respectively. The CE region is highly conservative among the three species. Regions of identity of three species are represented in red. Consensus or near-consensus binding motifs for factors implicated in muscle gene regulation (black boxes) were identified. d A statistical map of the regulatory region of myoD gene in pigs
Fig. 2. The mutation (Arg76Pro) of MyoD has no effect on its transcriptional activity. a Schematic representation of WT pig MyoD protein. Arrows denote amino acid positions of the mutation. b qPCR analysis for the expression of myoD in NIH3T3 cells treated with overexpression Flag-MyoDWTã€Flag-MyoDR76P or a control Flag vector. GAPDH was used as internal control. c Western blotting analysis for protein levels of MyoD by Flag antibody for 48 h in GM. d qPCR analysis was performed to detect the level of ckmã€cdh15 and myh3 at 3d post-differentiation. e 293 T cells were transiently transfected with 4Rtk-luc and expression vector encoding the indicated proteins. Cells were harvested 48 h post-transfection. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System Kit (Promega). The data was presented as the normalized ratio of Firefly luciferase activity to the Renilla luciferase activity. f The myotube formation was observed under white light after differentiation for 5 days. Scale bar =50 μm. g After transfection and differentiation for 6 days, MyHC was detected by immunofluorescence staining. Scale bar = 100 μm. Data are presented as mean ± S.E.M.; n = 3; *p < 0.05, ***p < 0.001 (Student’s t-test)
Fig. 3. There is no difference in the transcriptional activity of MyoD among four species. a Alignment of bHLH domain among mouse, pig, chicken and xenopus. b qPCR analysis for the expression of myoD in NIH3T3 cells treated with overexpression Flag-myoD plasmid of different species or a control Flag vector in GM for 36 h. c After transfection of 48 h, MyoD protein level was detected by Flag antibody. Ctrl represents a control group. (d, e) qPCR analysis was performed to detect the expression level of myog, ckm, cdh15 and myh3 in different species at 3d (d) or 6d (e) post-differentiation. f, g The significance analysis of expression levels of key factors of myogenic differentiation from D and E in different species to determine whether MyoD has the same transcriptional activity. h 293 T cells were transiently transfected with 4Rtk-luc and myoD expression vectors of different species. After transfection of 48 h, cells were harvested to measure Firefly and Renilla luciferase activities by Dual-Luciferase Reporter Assay System Kit (Promega). i Immunofluorescence detection of MyHC (green) in myoD plasmid of mouse, pig, chicken and xenopus or control transfected NIH3T3 cells after 7 days of differentiation. Data are presented as mean ± S.E.M.; n = 3; *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test); Scale bar =100 μm
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