| Body size traits are the most intuitive expression of phenotypic differences among animals,and also one of the key characteristics for identifying livestock breeds.The body size of livestock directly reflects its size,structure and development,and is closely related to its physiological function,production performance,disease resistance and adaptability to external environmental conditions.Body size is an important index to determine the quality of breeds and production performance.It has become a priority factor in livestock breeding.The size traits of horses are also very important,which is a key factor to determine the performance and economic value of horses.Chinese Debao pony is one of the most precious and naturally formed pony breeds in the world.It is of great significance to understand the key genes controlling the size traits of ponies and their regulation mechanism for revealing the genetic mechanism of dwarf traits of ponies.In this study,the short-statured Debao pony and the tall-statured Mongolian horse were selected as the research animals,and the pituitary and long bone tissues closely related to their physical development in juvenile and adult stages were selected.Through transcriptome sequencing and data analysis,the key genes for the formation of physical traits in horses were systematically screened.The correlation between the expression patterns of candidate genes and body size traits was largely revealed.It laid an important foundation for elucidating the molecular genetic mechanism of dwarfing characteristics of Debao pony.The results are as follows:(1)The results of transcriptome sequencing showed that there were 29243 differentially expressed genes in pituitary and epiphyseal tissues of juvenile and adult Debao ponies and Mongolian horses,including 13946 up-regulated genes and 15297 down-regulated genes.In adult horses,both pituitary and long bone tissues showed high expression level in Mongolian horse;while in juvenile horses,pituitary tissues showed high expression level in Mongolian horses;while long bone tissues showed high expression level in Debao pony.(2)It was found that the expression level of GH,TSHB and CGA genes in pituitary of Debao pony in juvenile was much higher than Mongolian horse,but there was no significant difference in the expression level of the above genes between in adult stages.Western Blot analysis showed that the pituitary GH expression level of Debao pony was higher than that of Mongolian horse in both juvenile and adult stages.The results of q-PCR were consistent with the data obtained by sequencing.(3)Further analysis of differentially expressed genes in the long bone tissues of the two horses showed that the expression of m-RAS,MAPK10 and ATF3 genes in GHR signaling pathway,which played an important role in bone growth,was significantly down-regulated in Debao pony data.While the expression level of PLC g 2 gene in WNT/Ca2+signaling pathway was significantly up-regulated.The expression level of TLR and its signaling pathway gene TGF ?3 was significantly up-regulated(p?0.05).These pathways can slow down the proliferation of osteocytes and lead to premature mineralization of extracellular matrix to eventually stop bone growth.It is presumed that the above three signaling pathways cooperate with each other and result in the formation of dwarf trait of Debao pony.(4)The expression level of WNT2,WNT ? PLC ?2 genes in the pathways was decreased by detecting the related genes in osteoblasts induced by horse bone marrow mesenchymal stem cells and ATDC5 cells after adding WNT pathway inhibitors.The expression level of GHR,m-RAS and ATF3 genes in the pathway was decreased by transfecting ATDC5 cells with lentivirus containing GHR-RNAi.The experimental results show that they are consistent with the analysis of sequencing data.(5)Plasma test showed that the concentration of IGF-1 was higher in juvenile Debao pony than that in adult,but the GH concentration was opposite.The plasma GH content of Mongolian horse in juvenile was higher than that of adult horse,but the concentration of IGF-1 was contrary,and the periods of GH and IGF-1 secretion were quite different between the two horses.These changes in hormone secretion were consistent with the trend of gene expression revealed by transcriptional sequencing analysis,which showed that Debao pony maintained the balance of its current body size in a body environment with severe GHR deficiency and excessive GH.In conclusion,the analysis of transcriptome mRNA levels in pituitary and epiphyseal tissues,which determine the key tissues of body height in Debao pony and Mongolian horse,showed that the expression patterns of GHR,IGF-1,TSHB and CGA,which are key candidate genes for regulating body height development,were significantly different between the two groups of horses,and the plasma content of major hormone proteins regulating body height growth and development tended to change with their gene expression.These key hormone genes were knocked down at the cellular level,and the expression changes of the related genes of signaling pathway were detected,and the results were consistent with expectations.The above comprehensive research results at gene,protein and cell levels have accumulated important data for revealing the molecular genetic mechanism of dwarf size formation in Debao pony,laid a solid foundation,provided key genetic resources for breeding special-purpose horses.At the same time,it provides a new research idea for revealing the genetic mechanism of human high-related diseases. |
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