| Antler is the young antlers of male deer that has not yet been ossified and have hairs,and are the only tissue organ in mammals that can periodically shed and completely regenerate.Antler is an ideal model for studying mammalian cell formation,growth and differentiation because it regenerates periodically and grows very fast.Understanding the function of growth factors in antler can provide scientific reference for tissue regeneration,wound repair and tumorigenesis.Sika deer is a typical representative of the deer family,and the molecular biology research on the growth and development of its antler is mostly focused on the mesenchymal layer of the apical tissue,while the research on other tissue layers such as antler tip skin and cartilage are relatively rare.The growth process of plum antler can be divided into three growth periods,namely,30 d,60 d and 90 d after dislocation,according to the typical characteristics of antler: small saddle,two bars and three branches.Studies have shown that the antler tip is the "growth center" of antler,and the network of genes that form the antler control all stages of antler growth in concert with each other,with complex and sophisticated internal molecular control mechanism.Therefore,it is of great scientific significance to study the expression of genes related to deer antler growth at different growth stages and their differences in expression for exploring the function and molecular regulation mechanism of each gene in the process of growth and development.The top tissue of deer antler is composed of antler tip skin,mesenchyme,precartilage and cartilage.In this study,the velvet skin tissue at the top of sika deer’s antler was used as the research material,and the KGF,FGF10,HDGF,NGF,MEF2 C,ATF3 genes which related to the growth and development of antler were extracted by extracting the total RNA of the antler tip skin,and obtained by RT-PCR and T-A cloning technology.According to the cloned cDNA sequence,the fluorescent quantitative primers were designed to explore the expression and differences of six growth-related genes in the early,middle and late stages of growth in the velvet skin tissue of sika derer antler-tip.The results were as follows:(1)The sequences with the complete cDNA coding regions of KGF,FGF10,HDGF,NGF,MEF2 C and ATF3 genes were successfully cloned and obtained,which with full lengths of 585 bp,642 bp,720 bp,726 bp,1302 bp and 546 bp,and encoded 194,213,239,241,433 and 181 amino acids.(2)The KGF,FGF10,and NGF genes encoded unstable hydrophilic proteins with transmembrane structures with relative molecular masses of 22,489.17,23,767.08,and36,628.48,respectively,and theoretical isopic PI values of 9.35,9.61,and 9.63.The secondary structure was dominated by random coils.The protein encoded by the HDGF gene is a stable hydrophilic protein with no transmembrane structure,the relative molecular mass of 26,534.94,the theoretical isoelectric p I of 4.70,and the secondary structure dominated by random coils.MEF2 C and ATF3 genes encode proteins that are unstable hydrophilic proteins without transmembrane structures.Their relative molecular mass was 46,898.44 and 20,565.60,respectively.Theoretical isoelectric p I values were 8.69 and 8.58,respectively.The secondary structure was dominated by random coils and α-helix.(3)The Blastp comparison showed that the six growth-related genes in this study are highly conserved in the evolution of the antler,with little interspecific variation.The cDNA sequences of the genes are all more than 95% similar to those of homologous species such as white-tailed deer,cattle and sheep,and the amino acid sequences of the encoded proteins are more than 96% similar to those of homologous species such as white-tailed deer,cattle and sheep.It is hypothesized that these six growth-related genes have important functions in the evolution of the species and are key functional genes in life activities,which may have important regulatory roles in antler regeneration and growth development.(4)The six growth-related genes in this study were expressed in each growth stage of sika deer antler skin tissue,and the expression levels were different in different stages.The expression of KGF gene was significantly different between the early stage and the middle stage,the early stage and the late stage(P<0.05),and the difference between the middle stage and the late stage was not significant(P>0.05).The middle stage is 2.1585±0.0141 times of the early stage,and the latter stage is 1.7285±0.2253 times of the early stage;The expression levels of FGF10 gene were significantly different between the early stage and the middle stage,the early stage and the late stage,and the middle stage and the late stage(P<0.01).The middle stage is 0.0947±0.0087 times of the early stage,and the latter stage is 2.3897±0.0468 times of the early stage;There was no significant difference in the expression of HDGF gene between the early stage and the middle stage(P>0.05),the expression level between the early stage and the later stage was extremely significant(P<0.01),and the expression level between the middle stage and the later stage was significantly different(P<0.05).The middle stage is1.3258±0.2985 times of the early stage,and the latter stage is 3.7235±0.5319 times of the early stage;The expression levels of NGF gene were significantly different between the early stage and the middle stage,the middle stage and the later stage(P<0.05),and the expression levels of the early stage and the later stage were significantly different(P<0.01).The middle stage is1.9334±0.2143 times of the early stage,and the latter stage is 3.2851±0.1663 times of the early stage;The expression of MEF2 C gene was significantly different between the early stage and the middle stage,the early stage and the late stage(P<0.01),and the expression level of the middle stage and the late stage was not significantly different(P>0.05).The middle stage is3.2106±0.1835 times of the early stage,and the latter stage is 3.6201±0.1951 times of the early stage;The expression level of ATF3 gene was significantly different between the early stage and the middle stage,the early stage and the later stage(P<0.01),and the expression level of the middle stage and the later stage was significantly different(P<0.05).The middle stage is3.0835±0.1813 times of the early stage,and the latter stage is 5.5429±0.4433 times of the early stage.In conclusion,this study found that the KGF,FGF10,HDGF,NGF,MEF2 C and ATF3 genes are highly conserved in evolution and have important roles in the growth and tissue repair of antler,with differences in the relative expression in antler skin tissues at different times.This study lays the foundation for further exploration of the molecular mechanism of antler growth and provides a reference for tissue injury repair in mammals. |
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