Cloning of NGF Gene in Red Steppe Cattle,and Its Expression in Myogenic Differentiation of Skeletal Muscle Satellite Cells

doi:10.7668/hbnxb.20196603

Abstract

Abstract:

In order to explore the gene function of nerve growth factor(NGF),and to clarify the expression differences of NGF in different tissues of Red Steppe cattle and its influence on the myogenic differentiation of bovine skeletal muscle satellite cells(bSMSCs),collect various tissues of the Red Steppe cattle,clone and obtain the complete CDS region of the NGF gene,and then conduct bioinformatics and tissue expression analysis on it.bSMSCs were isolated from the deep tissues of the hind limbs of neonatal calves.The expression of Pax7,Desmin and MyoD1,the specific markers of bSMSCs,was detected by immunofluorescence staining.In vitro,2% horse serum was used to induce myogenic differentiation of bSMSCs by serum deprivation method,and the expression patterns of NGF mRNA and protein at different stages of differentiation were detected.The results showed that the length of the CDS region of bovine NGF gene was 726 bp,encoding 241 amino acids.The amino acid sequence of Red Steppe NGF gene is located in a branch of house mouse,red deer and sheep,with high homology and close kinship.The molecular weight of NGF protein was about 26.67 ku,the molecular formula was C₁₁₇₉H₁₈₆₁N₃₄₉O₃₃₉S₁₀,the theoretical isoelectric point was 9.72,the half-life was 30 h,and the NGF protein was water soluble.NGF protein had signal peptide and was secreted protein.There were 42 potential phosphorylation sites.There were three potential sites for N-glycosylation.The secondary structure of NGF protein was composed of α-helix,β-sheet,β-turn and random coil,and the results of tertiary structure prediction were consistent with those of NGF protein.There were significant differences in the expression level of NGF gene in different tissues,with the highest expression level in liver,followed by kidney and tumour.The expression of the NGF gene was continuously upregulated during the myogenic differentiation process of bSMSCs,and there was a significant difference on the 7th day.This study successfully cloned the CDS region sequence of the NGF gene in the grassland,and established the tissue expression profile of the NGF gene in the Red Steppe cattle.The expression pattern of the NGF gene during the myogenic differentiation process of bSMSCs was clarified.

Key words: Red Steppe cattle, NGF gene, Clone, Differentiation expression, Bioinformatics, Myogenic differentiation

CLC Number:

LI Xin, LI Changhong, LIU Yu, CAO Yang, FANG Wenwen, XIAO Cheng, CAO Yang. Cloning of NGF Gene in Red Steppe Cattle,and Its Expression in Myogenic Differentiation of Skeletal Muscle Satellite Cells[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(4): 222-230. doi: 10.7668/hbnxb.20196603.

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Figures/Tables 16

Tab.1 Primer sequences

基因 Genes	用途 Application	引物序列(5'—3') Primer sequence(5'—3')	片段大小/ bp Product size	退火温度/℃ Annealing temperature
NGF	PCR	F:ATGTCCATGTTGTTCTACACTCTGA R:TCAGGCTCTCTGCCCAGTC	726	55
NGF	qRT-PCR	F:ACATCAAGGGCAAGGAGGTG R:CAGTCTTCCTGCTGAGCACA	243	60
GAPDH	qRT-PCR	F:GTCGGAGTGAACGGATTCGG R:CCAGCATCACCCCACTTGAT	238	60

Tab.2 Bioinformatics analysis tools

项目 Item	分析工具 Analysis tool
蛋白理化特性	ExPASy ProtParam
Physicochemical properties of proteins
亲/疏水性预测	ProtScale
Hydrophilicity/hydrophobicity prediction
蛋白质磷酸化分析	Target P
Protein phosphorylation analysis
信号肽预测	SignalP 4.1 Server
Signal peptide prediction
蛋白质磷酸化位点预测	NetPhos 2.0 Server
Protein phosphorylation site prediction
蛋白质二级结构预测	NOVOPRO
Protein secondary structure prediction
蛋白质三级结构预测	SWISS-MODEL
Protein tertiary structure prediction

Fig.1 PCR amplification product from the CDS region of the NGF gene

Fig.2 Homology comparison of NGF gene sequences

Fig.3 Construction of the phylogenetic tree of NGF gene sequences

Tab.3 Amino acid composition of NGF protein

氨基酸 Amino acid	数量 Amount	频率/% Frequency	氨基酸 Amino acid	数量 Amount	频率/% Frequency
Ala(A)	23	9.5	Met(M)	4	1.7
Arg(R)	17	7.1	Phe(F)	12	5.0
Asn(N)	8	3.3	Pro(P)	13	5.4
Asp(D)	33	13.6	Ser(S)	19	7.9
Cys(C)	6	2.5	Thr(T)	20	8.3
Gln(Q)	9	3.7	Trp(W)	4	1.7
Gly(G)	14	5.8	Tyr(Y)	3	1.2
His(H)	11	4.6	Val(V)	18	7.5
Ile(I)	13	5.4	Pyl(O)	0	0.0
Lys(K)	14	5.8	Sec(U)	0	0.0

Fig.4 Prediction of the hydrophilic/hydrophobic nature of NGF protein

Fig.5 Prediction of phosphorylation sites of NGF protein

Fig.6 Prediction of N-glycosylation sites of bovine NGF protein

Fig.7 Prediction of the signal peptide of NGF protein

Fig.8 Prediction of the secondary structure of NGF protein Red.α-helix;Blue.β-sheet;Black.β-turn and random coil.

Fig.9 Prediction of the tertiary structure of NGF protein

Fig.10 Expression of the NGF gene in different tissues of the Red Steppe cattle Compared with the heart tissue,different capital letters indicate extremely significant differences(P<0.001).The same as Fig.13.

Fig.11 Isolation and identification of bSMSCs A.Primary bSMSCs cultured for 2 days;B.Primary bSMSCs cultured for 5—7 days; C.The third generation of bSMSCs; D.Immunofluorescence staining of specific marker Pax7 for bSMSCs; E.Immunofluorescence staining of specific marker Desmin for bSMSCs; F.Immunofluorescence staining of specific marker MyoD1 for bSMSCs.

Fig.12 Immunofluorescence staining of MyHC in bSMSCs on the 7th day

Fig.13 Role of NGF in the regulation of myogenic differentiation A.NGF mRNA expression on days 0,3,and 7;B.NGF protein expression on days 0,3,and 7.

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