| The ras superfamily is a kind of important functional protein, they mediate growth factors, cytokines and a variety of extracellular signal channels and play a greaat role in cells growth, differentiation, survival, proliferation, etc. Members of the Ras superfamily RERG (Ras-related, estrogen-regulated, growth-inhibitory) is the molecular switches of GTP/GDP and the growth inhibitory activity genes; Transforming growth factor-? is a set of superfamily of regulating cells growth and differentiation, TGF-? type ? receptor (transforming gowth factor preeeptorl TGF beta R1/TGFBRl) plays a negative regulation role in cells growth through the TGF-beta/ Smad signal pathway. This experiment selected RERG and TGFBR1 genes as the candidate genes for growth and a total of 978 Iraq rabbits,New Zealand rabbits, egger rabbits and tianfu black rabbits were selected randomly as trial materials. SNPs loci of two gene encoding sequence were identified by the direct sequencing method and divided into different genotypes via HRM technology and then analysed the correlation of the candidate SNPs with the growth of rabbits. Subsequently, the newborn sons of New Zealand rabbit were select ed as the experimental materials, Skeletal muscle satellite cells (Skeletal muscle satellite cells, SCs) were separated and purified by using the enzyme digestion method combining percoll discontinuous density gradient centrifugation. And siRNA was used to mediate TGFBR1 gene silencing expression in order to study the influence of the SCs proliferation of the rabbit. The main results were as follows:1.3 synonymous mutations were detected on RERG gene exon 5, respectively is c.255 T> a. c.264 G> A, c.330 G> A, which were complete linkage.Aiming at c. 330 G> A locus,423 rabbits(tianfu black rabbit 183, New Zealand 120, egger 65, Iraq 55) were divided into different genotype by using HRM technology and were studyed on correlation analysis. The results showed that the weight and daily gain during 42-84 days of AA genotype of RERG gene is significantly higher than that of AG genotype; AA genotype of IRA rabbit 84 days-weight is significantly higher than that of GG genotype individuals; AA genotype individuals of New Zealand rabbit average daily gain of 42-84 days of age is significantly higher than that of GG genotype.2. Detects an SNP locus were detected on the TGFBR1 gene exon 8,that is, c. 1264 c> A of which amino acids codon tranformed fromCGG to AGG and this mutation belonged to synonymous mutation. Only CC and CA genotypes were detected. On c.1264 locus 555 rabbits (tianfu black rabbit 206, New Zealand 188, Edgar,161) were dvided into different genotypes via HRM technology and susequently analysed correlation between the gene and the growth of rabbits. The results showed that AA genotype individuals of Edgar rabbits had significant higher 56 days of age weight than AC genotype individuals;70 days of age weights of AA genotype individuals of Tianfu black rabbit and New Zealand rabbits was significantly higher than AC genotype individuals;the average daily gain of 42-84 days and of 84 days of age of AA genotype individuals of the three varieties of rabbits were significantly higher than that of AC genotype.3. Newborn from New Zealand white rabbits were Selected and of which hind leg muscle tissue was separated and then SCs were purified. Through morphology observation and immunohistochemical detection, mark protein Desmin was detected,and we found that Desmin was positive expression in cells. This showed that the trial for the separation and purification of rabbit skeletal muscle satellite cells was succeed, and the purity of the rabbit skeletal muscle satellite satisfied the demands the follow study.4. Aiming at TGFBR1 gene, three specific siRNA sequences were designed and transfected SCs. TGFBR1 gene mRNA was detected by q-PCR, and the best siRNA is si-TGFBR1-01 of which the best transfection concentration was 50 nmol/L si-TGFBR1-01.Finally cells after 48 h transfected exibited that the mRNA expression level of inhibition efficiency of target genes was 87.61%.5. mRNA expression level of TGFBRl gene in cells decresed After transfected si-TGFBR1-01combining with downstream gene of TGF-?/Smad signaling pathways, Smad3, also had a decrease somehow.But the mRNA expression of TGFB1 gene which was the ligand gene of TGFBR1 appeared to rose.mRNA expression of MSTN gene ruduced either.The results showed that TGFBR1 gene through the TGF-?/Smad signaling pathways played a negative regulatory role on skeletal muscle cells proliferation.6. As for the transfection experiments of SCs cells at logarithmic phase,the effects on cells proliferation of the 50 nmol/L si-TGFBR1-01 transfection after 48 h was detected. The trial showed that after TGFBRl gene mRNA expression was suppressed, cells' proliferation capacity were significantly higher than the control group (P<0.05) which suggested that TGFBR1 negatively regulated the proliferation of skeletal muscle cells.The SNPs on CDS of RERG and TGFBR1 genes of rabbits were in association with growth speed significantly.RFRG and TGFBR1 gene can be used as important candidate genes for the growth traits of rabbits.Through the trail on inhibition of TGFBR1 gene expression that can considered that TGFBR1 gene played negative a role of regulation on skeletal muscle cells proliferation. This study had provides theory basis for further study of TGFBR1 gene or TGF-?/Smad signaling pathways in growth and development of skeletal muscle cells. |
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