The Funtion Of SMAD5 Gene In The Proliferation And Differentiation Of Bovine Myoblast And Associations With Meat Quality Traits In Qinchuan Cattle

The number of postnatal muscle fibers remains stable,thus muscle fiber hypertrophy is the main determinant of the increase in skeletal muscle mass,ultimately affecting meat quality and flavor.SMAD family proteins(SMAD 1-8)are a class of important cell signal transduction proteins,which directly involved in signal transduction with TGF-?superfamily members.Previous studies explored important positive regulatory function of SMAD1,SMAD5 and SMAD8 in skeletal muscle mass through BMP signaling pathway,and 80%reduction in the myofibrils size was reported by knockdown of these genes.Notably,recent studies have confirmed the theory that BMP-SMAD1/5/8 signaling significantly promotes muscle mass by outcompeting the binding to SMAD4 with pathway of activin/myostatin/TGF?which strongly inhibits myocyte differentiation.However,studies on bovine SMAD5 gene in myoblasts have not been reported.Therefore,by isolating and culturing primary Qinchuan cattle myoblasts,this study explored the role and mechanism of the SMAD5 gene in regulation of bovine myoblasts proliferation and differentiation.The main results are shown as follows:(1)Tissue expression patterns of SMAD5 gene in Qinchuan cattle.The expression of SMAD5 gene was ubiquitous,and the highest of that was in the omasum,large intestine and longissimus dorsi.In the longissimus dorsi of Qinchuan cattle,the m RNA levels of SMAD5gene changed with the growth:it first increased,then decreased significantly after maintaining a high level at 6 and 12 month of age(P?0.01),and finally increased slowly after18 months.(2)Localization and expression of SMAD5 gene in myoblasts.The subcellular localization indicated that bovine SMAD5 protein was expressed both in the nucleus and cytoplasm.In the process of myoblast induced differentiation,the m RNA of SMAD5 gene significantly increased in the first two days of induction(P?0.01),and then slowly increased(P?0.05).(3)Silencing SMAD5 gene inhibited the proliferation and differentiation of myoblasts.A piece of si RNA,namely si SMAD5,was obtained to effectively silence SMAD5 gene as?80%in Qinchuan bovine myoblasts.si SMAD5 significantly increased the cell counts in G2/M phase(P?0.01),but had no effect on the number of cells in G0,G1 and S phase(P?0.05).In addition,both the m RNA and protein levels of CDK1 decreased with the down-regulation of SMAD5(P?0.01),while CDK2 showed no significant changes(P?0.05).Compared with the control group,myoblasts that down-regulated SMAD5 gene were significantly impaired in the process of muscle differentiation,and the m RNA level of Myo G was also significantly decreased(P?0.01).(4)The transcriptional level of SMAD5 gene was decreased by the silence of transcription factors Fox P1 and OSR1,which inhibited myoblast differentiation.5?RACE and sequence alignments estimated transcription initiation site of Qinchuan cattle SMAD5 gene,the adenine residue(A)which was defined as+1,was fully consistent with the its published m RNA sequence in NCBI database.Analysis of dual-luciferase activity revealed that the core region of SMAD5 gene was located at region of-603 bp?-231 bp.Site-directed mutation assays,RNAi technology and EMSA assays in vitro indicated that Fox P1 and OSR1positively regulated the transcriptional activity of SMAD5 gene via binding to the core promoter.In addition,silencing of Fox P1 and OSR1 genes in bovine myoblasts sharply reduced m RNA expression of SMAD5 and myogenic regulatory factor(MRFs)(P?0.01).(5)The diplotype H₁-H₃ of SMAD5 gene was the dominant haplotype combination type of back fat thickness(BT)and ultrasound loin muscle area(ULA)of the Qinchuan beef cattle.It was detected 3 SNP sites,which is AC?000164.1:g.A-1593 A?G(SNP1),g.-953 C?T(SNP2),g.-1226 C?T(SNP3),and eight haplotypes(Hap₁-Hap₈)in SMAD5 promoter region of 448 Qinchuan beef cattle.Hap₁(-ACC-,46.2%),Hap₂(-ACT-,20.3%)and Hap₃(-ATC-,19.9%)had higher frequencies.Analysis of enzyme activities of luciferase reporter vectors showed that transcriptional activities of SMAD5 promoters containing haplotypes Hap₃ and Hap₄ were significantly higher than that of other haplotypes(P?0.01).Correlation analysis of5 diplotypes of SMAD5 gene and the production traits of Qinchuan beef cattle showed that compared with other diplotypes,individuals with diplotypes of H₁-H₃ generally had higher back fat thickness(BT)and ultrasound loin muscle area(ULA)(P?0.01),while there was no significant effect on body measurement traits(BMTs)and intramuscular fat content(IFC)(P?0.05).In conclusion,SMAD5 gene plays an important role in the muscle growth and development of Qinchuan cattle.Silencing SMAD5 inhibits myoblast proliferation by down-regulating CDK1 expression and delaying cell cycle progression.Knockdown the transcription factors Fox P1 and OSR1 in myoblasts inhibits the transcriptional activity of SMAD5,thereby inhibiting myoblast fusion to form myotubes.Furthermore,In addition,the diplotype H₁-H₃ of SMAD5 gene identified by correlation analysis was an effective molecular marker for the early selection of Qinchuan beef cattle with excellent meat quality traits.This study preliminarily revealed the regulatory mechanism of SMAD5 gene in the muscle formation process of Qinchuan cattle,providing new scientific data for beef cattle breeding in China.