The Effect of Down-regulation of BPGM on the Growth and Development of Myogenic Cells in Jingyuan Chickens

doi:10.7668/hbnxb.20194625

Abstract

Abstract:

The aim of this study was to investigate the effect of bisphosphoglycerate mutase(BPGM)on the growth and development of chicken myogenic cells.The transcriptomics and proteomics data was jointly analyzed in the preliminary stage of the study to identify the differentially expressed genes that might affect the growth and development,and investigated their effects on the proliferation,apoptosis and expression levels of related marker genes of myogenic cells.Inosinic acid(IMP)was detected by ELISA and adenosine triphosphate(ATP)was detected by colorimetric assay,which revealed the important roles of the key candidate genes on muscle development and meat flavor of Jingyuan chickens.The results showed that a total of 139 differentially expressed genes/proteins were identified between the breast muscle and leg muscle of Jingyuan chickens,including 75 differentially expressed genes/proteins that were up-regulated in both the transcriptome and proteome,45 differentially expressed genes/proteins that were down-regulated in both the transcriptome and proteome,13 differentially expressed genes/proteins that were down-regulated in the transcriptome,and 6 differentially expressed genes/proteins that were up-regulated in the proteome and down-regulated in the transcriptome.7 differentially expressed genes were screened from the glycolysis/glycolysis pathway,which were BPGM, phosphoglycerate kinase 1 gene(PGK1),glucose-6-phosphate isomerase gene(GPI),phosphoglycerate mutase 1 gene(PGAM1),lactate dehydrogenase A gene(LDHA),lactate dehydrogenase B gene(LDHB)and phosphoglycerate isomerase 1 gene(TPI1),and screened for the differentially expressed gene BPGM in combination with KEGG pathway enrichment analysis and protein network interactions analysis.BPGM was expressed in all tissues of Jingyuan hens,the highest expression was in pectoral muscle,and it showed an up-regulation trend during the differentiation of myoblasts.Primary myogenesis cells were isolated and cultured for cell transfection.Interfering with BPGM inhibited myoblast proliferation and promoted apoptosis;it also affected genes upstream and downstream of the glycolytic pathway,and inhibited the biosynthesis of IMP and ATP at the same time.In conclusion,down-regulation of BPGM inhibited the growth and development of myoblasts,as well as the biosynthesis of IMP and ATP.

Key words: Jingyuan chicken, BPGM, Glycolysis, Myogenic cells, Growth and development, Meat flavor

FU Xi, ZHAO Wei, YU Baojun, ZHANG Tong, CHEN Yafei, MAO Xiangyuan, WEI Chuanhao, MA Xiaoping, ZHANG Xinyu, ZHANG Juan. The Effect of Down-regulation of BPGM on the Growth and Development of Myogenic Cells in Jingyuan Chickens[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(2): 219-230. doi: 10.7668/hbnxb.20194625.

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Figures/Tables 8

Tab.1 si-BPGM target sequences

基因 Gene	序列名称 Sequence name	靶序列 Target sequence
BPGM	si-BPGM-1	GGCATCTCTGACAAGGACA
	si-BPGM-2	CAATGACCGTCGATACAAA
	si-BPGM-3	GGCTCTGAATCATGGAGAA

Tab.2 The RT-qPCR primers information

基因 Gene	登录号 Accession No.	序列(5'—3') Sequence(5'—3')	产物长度/bp Product length
ACTB	L08165.1	F:ATGGACTCTGGTGATGGTGTTAC	185
		R:TCGGCTGTGGTGGTGAAG
BPGM	NM_001030768.2	F:GTGCTTGATAGACTCCTTCC	135
		R:CTTGTCAGAGATGCCTTCC
Cyclin E	BI390175	F:GGTTGTTGGCATCAGTAAAG	214
		R:TATTACCTCATCTTCCTCGTTG
Cyclin E	NM_205381.2	F:CAGAAGTGCGAAGAGGAAGT	189
		R:GATGGAGTTGTCGGTGTAAAT
PCNA	NM_204170.3	F:GACTCCTCGCACGTCTCCCT	161
		R:TCCGCATTGTCTTCTGCTCTG
CDK1	NM_205314.2	F:TGCAAGGCTGTACCTCATT	174
		R:ACTCTTAACACGCGAACGA
BAK1	NM_001030920.3	F:GTTCGACAGCGGCATTA	158
		R:TGCCTTGCTGGTAGACG
FAS	NM_001199487.2	F:GTTCGTCATCACCGTCTATCG	215
		R:TTCGTAGGCTCCTCCCATC
BID	NM_204552.3	F:TCACAGGCAGTGGAAGGA	196
		R:TGTGGAAGTGTTGGCTGAT
Caspase9	BG711928	F:CGAAGGAGCAAGCACGA	187
		R:AGGTTGGACTGGGATGGAC
PGM1	NM_001038693.3	F:AACCACCTCAAGATTCGC	212
		R:GCAGCTCCAAAGTCGTAC
PFKM	NM_204223.2	F:TGGCTGTGCTCAATGTGG	244
		R:GTGGCGCTGATTTCCTC
TPI1	NM_205451.1	F:GAAGATGAACGGCGACAA	236
		R:CCCATGCAGCTCCAATAT
PGK1	NM_204985.4	F:GATGTGAAGGGCAAGCG	279
		R:CTCCACCTCAGGACCAA
GAPDH	NM_204305.2	F:TGCCGTCTGGAGAAACC	160
		R:CAGCACCCGCATCAAAG
ENO2	NM_204876.2	F:TCCTGCTCAAAGTCAACC	123
		R:TGAAAGTGTCTTCGGTCTC
ENO3	NM_205119.1	F:TGGACGGCACCGAGAAT	155
		R:CGGGACAGGCAGGATGA
PKM2	NM_205469.2	F:CACGGCACCCACGAGTA	210
		R:GGCATTGTCCAGCGTCA

Fig.1 Joint transcript and protein analysis and differential gene screening A.Nine-quadrant plot of total differentially expressed gene/protein correspondences:Cor-up.Red dots indicate transcriptional and protein co-regulation;Cor-down.Green dots indicate transcriptional and protein co-regulation;Opposite-sig.Yellow dots indicate transcriptional up-regulation but protein down-regulation,blue dots indicate transcriptional down-regulation but protein up-regulation;B.KEGG pathways enriched by differentially expressed genes/proteins;C.GO entries enriched by differentially expressed genes/proteins;D.Protein network interactions of differentially expressed genes/proteins BPGMs;E.G/G pathway:Purple pathway for glycolysis,red pathway for gluconeogenesis,green for the location of BPGM.

Fig.2 Expression analysis of chicken BPGM gene A.Expression analysis of BPGM gene in hen tissues;B.Expression analysis of BPGM gene in differentiation of adult myoblasts.Different lowercase letters indicate significant differences(P<0.05)and different uppercase letters indicate highly significant differences(P<0.01).The same as Fig.3—6.

Fig.3 Immunofluorescence identification of myofibroblasts and detection of cellular interference efficiency A.Chicken primary myoblasts on day 1—3;B.Immunofluorescence identification of F₁ generation myoblasts;C.Fluorescence of si-BPGM transfected at 50 nmol/L;D.Transfection efficiency of si-BPGM with different concentrations of different interference sequences.

Fig.4 Effects of interfering with BPGM on the proliferation of adult myoblasts A.EdU assay to detect the effect of interference with BPGM on the proliferation of myoblasts;B.Percentage of EdU-positive cells;C.CCK-8 assay to detect the effect of interference with BPGM on the proliferation of myoblasts at different time periods after the interference with BPGM;D.Transfection for 48 h to detect the expression of myoblasts proliferation marker genes.

Fig.5 Effect of interfering BPGM on apoptosis of myoblasts A.Effect of interference with BPGM on apoptosis of myoblasts detected by Annexin V-mCherry/SYTOX Green; B.Percentage of SYTOX Green-positive cells;C.Expression of apoptosis marker genes in myoblasts detected by transfection for 48 h.

Fig.6 Effects of interfering with BPGM on glycolysis and the content of IMP and ATP in myoblasts A.Expression of upstream and downstream genes in the G/G pathway after interfering with the BPGM gene; B.The effect of interfering with BPGM on IMP content in adult myocytes;C.The effect of interfering with BPGM on ATP content in adult myocytes.

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