| Animal growth and development were mainly regulated by GH-GHR-IGF-?axis,in which the growth hormone receptor(GHR)played a central role.Except IGF-I depended pathway,GHR can also act directly on the target organs to regulate the sugar,lipid and protein metabolism process.Thus it took a significant part in animal growth,morphogenesis and maintaining body steady state from the levels of organization system.In order to verify the relationship between pGHR including its regulation and the trait of body size in pig,we choose Bama-mini pig as our research object owing to its dwarf body size,and select Landrace pig,a large body size breed,as control,to do the comparative study on such aspects as serum GHR levels,gene coding sequence SNP analysis,gene expression and regulation of its transcription.The researching results were shown as follows:1.In Landrace the serum levels of GHR and IGF-I were significantly higher than those in Guangxi Bama-mini pig on day 1,180 of age(P<0.01).However,on day 30 the differences were not significant(P>0.05).The serum content of IGF-I and GHR had the same changing trends in both pig breeds.The serum of GH kept relative constant levels in different ages,and no significant difference in serum GH level was detected in the two different breeds(P>0.05).2.The cloned Bama-mini pig GHR cDNA contained 6 SNPs,in which two of them are missense mutation lead to A1225S and V1801I,others were synonymous mutations.The Bama-mini pig and Landrace GHR over expression vectors were constructed and used to transfected PK15 cells,and then the expression of JAK2 gene in the transfected PK15 cells was detected by QRT-PCR.It was found that the expression level of JAK2 gene was significantly higher in the cells transfected with Landrace GHR over expression vector(P<0.01).The result indicated that the difference in CDS sequences of GHR between Bama-mini pig and Landrace may cause different regulation function to its downstream gene.3.Two pGHR SNPs(A1248G and A1801G)loci were verified in F2 generation of "Bama×Landrace" resources population,and the correlation between these SNPs loci with growth traits was determined.The results showed that 2 months old pigs with 1248GG and 1248AG genotypes had larger chest circumference compared to 1248AA genotype and 4 months old pigs with 1248GG and 1248AG genotypes had heavier body weight(P<0.05).The 1801GG and 1801 AG genotypes were also found having lager chest circumference in 3 and 6 months of age.However,the highest withers height in 1 and 6 months of age was found in pigs with 1801AA genotype(P<0.05).Two loci haplotype analysis found that in 1 months old pig,withers height of-AAAA-and-AGAG-genotype was higher compaired with-GGGG-genotypes,heavier body weight in 4 months of age was found in-GGGG-genotypes compaired with-AAAA-and-AGAG-genotype and chest circumference of 6 months old pig was larger in-GGGG-genotypes compaired to-AAAA-and-AGAG-genotype(P<0.05).4.In Bama-mini pig and Landrace QRT-PCR was used to analyze the expression of GHR and IGF-I on day 1,180 of age.The expression of GHR gene was detected in tissues including heart,liver,muscle,spleen,kidney and brain.It was found that the expression of GHR and IGF-I showed different levels owing to different pig breeds.The expression level of GHR and IGF-I were significantly higher in liver and muscle from Landrace on 180d of age(P<0.05).Thus,it was speculated that the low expression level of GHR may be related to the slower growth rate and dwarf body size of Bama-mini pig.5.The sequence of cloned mRNA 5'-1A,5'-1B and 3' cDNA sequence of GHR showed no differences between Bama-mini pig and Landrace.However,Bama-mini pig showed lower expression level of GHR 5'-1A than Landrace in liver on day 180 of age.Segments of 2700 and 2696 bp from ATG upstream were obtained by segmented amplification of GHR promoter region in Bama-mini pig and Landrace respectively.Sequence analysis of the obtained GHR promoter region from Bama-mini pig found that there are 3 bp deletion mutation in loci 733-735 bp and 7 bp insertion mutation in loci 1909-1915 bp.In addition,several SNPs were also found in the GHR promoter region in Bama-mini pig.The deletion and insertion mutation and other SNPs found may created different combination sites for relevant transcription factors,in this way they could influence expression and transcriptional regulation in GHR and related genes.Bama-GHR-P1-EGFP and Bama-GHR-P2-EGFP that the active detection vectors involved in GHR gene promoter region were constructed and used to transfected PK15 cells,The fluorescence detection results indicated that the transcription of GHR gene could be activated by 1507 bp and 1603 bp fragments involved in the promoter region.The CpG island in GHR gene promoter region also showed different methylation levels between Bama-mini pig and Landrace.The methylation level in CpG island in GHR gene promoter region from liver of Bama-mini pig was significantly higher on day 180 of age(P<0.01).It was thus suggested that methylation level in GHR gene promoter region may be involved with the liver GHR gene transcription regulation. |
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