| Antler is a bony organ that can be periodically shed and completely regenerated in deer.Its regeneration and growth are ideal experimental animal models for mammalian developmental biology and human biomedical research.Antler grows rapidly at a rate of about2cm/d in the growth period,without the unique biological characteristics of "cancer",which has attracted much attention.Many studies have shown that antler growth is controlled by the antler apical tissue(the growth center),where growth factors and transcription factors cooperate to form a complex regulatory network.At present,the study on the expression pattern of growth factors,transcription factors and other related genes in the growth of antler is one of the hot topics in the research on the growth and development of antler.Based on this,in this study,six important genes related to the growth of antler were selected for each tissue layer at the tip of antler during its growth process: Osteopontin(OPN),Parathyroid hormone like hormone(PTHLH),Stathmin2(STMN2),Midkine(MDK),MAF b ZIP transcription factor K(MAFK),Basic transcription factor 3 like 4(BTF3L4).In order to reveal the internal molecular mechanism of rapid growth regulation of antler and provide new ideas for tissue wound repair and organ regeneration in mammals,we studied the expression and regulation of growthrelated genes at the tip tissue layers of antler during growth from the perspective of time and space.In this study,three 5-year-old healthy adult male sika deer were selected,and tip tissues were collected during the early(antler with a saddle-like appearance),middle(antler with two branches),and late(ntler with three branches)periods of antler growth.In other words,the dermis,mesenchyme,precartilage,cartilage tissue layers from the antler tip were collected at about 30,60 and 90 days after the antler shed.To study the temporal and spatial expression differences of OPN,PTHLH,STMN2,MDK,MAFK,BTF3L4 genes closely related to antler growth.The cDNA sequences of the growth-related genes of the deer antler were obtained by molecular cloning technology.The physicochemical properties,subcellular localization,hydrophilicity and hydrophobicity,transmembrane structure and other biological information of the proteins encoded of each gene were mined and analyzed by bioinformatics technology.The temporal and spatial expression differences of genes in the tip tissues of antler were investigated by real-time quantitative PCR.The results are as follows:1.The cDNA sequences of OPN,PTHLH,STMN2,MDK,MAFK,BTF3L4 genes of sika deer were successfully cloned and obtained,and the length of which were 962 bp,610 bp,630 bp,528 bp,503 bp and 525 bp,respectively.Each gene contained the complete coding region sequence,the coding region sequence length was 843 bp,534 bp,540 bp,429 bp,474 bp,477 bp,encoding 280,177,179,142,157,158 amino acids,respectively.2.Bioinformatics analysis revealed that the protein encoded of OPN gene was an unstable hydrophilic protein localized outside the cell without transmembrane structure.The protein encoded of the PTHLH,STMN2,MAFK genes were unstable hydrophilic protein localized in the nucleus without transmembrane structure.The protein encoded of MDK gene was a stable hydrophilic protein with transmembrane structure localized in the nucleus.The protein encoded of BTF3L4 gene was a stable hydrophilic protein localized in the nucleus without transmembrane structure.3.By comparing multiple sequences,it was found that the growth-related genes of antler were relatively conserved in evolution and had little difference from other species.The similarity of amino acid sequence of encoded protein to corresponding protein of homologous species such as cattle and sheep was more than 90%.It is speculated that they are functional genes necessary to maintain normal life activities and may regulate the rapid growth of antler.4.Real-time quantitative analysis showed that growth-related genes were expressed in all tissues of antler tip at different growth periods,and there were temporal and spatial expression differences:(1)In the dermis tissue,the expression levels of PTHLH,MDK,BTF3L4 gene were extremely significantly higher in the middle period than those in the early period(P<0.01),and extremely significantly lower in the late period than those in the middle period(P<0.01).The expression levels of STMN2,MAFK gene in the middle period were extremely significantly lower than those in the early period(P<0.01),and the expression levels in the late period were extremely significantly lower than those in the middle period(P<0.01).(2)In the mesenchyme tissue,the expression levels of OPN,PTHLH,BTF3L4 gene were continuously up-regulated,and the expression levels in the late period were significantly higher than those in the early period(P<0.05).The expression levels of STMN2,MDK,MAFK gene in the middle period were extremely significantly higher than those in the early period(P<0.01),and the expression levels in the late period were significantly lower than those in the middle period(P<0.05).(3)In the precartilage tissue,the expression levels of OPN,STMN2,MDK gene were upregulated continuously,and the expression levels in the late period were significantly higher than those in the early period(P<0.05).The expression levels of PTHLH,BTF3L4 gene were extremely significantly higher in the middle period than in the early period(P<0.01),and extremely significantly lower in the late period than in the middle period(P<0.01).(4)In the cartilage tissue,the expression levels of OPN,BTF3L4 gene were continuously up-regulated,and the expression levels in the late period were extremely significantly higher than those in the middle period(P<0.01).The expression levels of PTHLH,STMN2,MDK gene in the middle period were extremely significantly higher than those in the early period(P<0.01),and the expression levels in the late period were extremely significantly lower than those in the middle period(P<0.01).(5)In the early period,the expression of OPN,PTHLH,STMN2,MDK,MAFK,BTF3L4 gene in mesenchyme tissue was 1.2537±0.0981,0.2368±0.0248,0.0556±0.0076,0.0621±0.0100,0.0777±0.0157,2.0978±0.3365 times of that in dermis tissue,respectively.The expression in precartilage was 5.6864±0.3627,3.5732±0.4091,0.1625±0.0167,0.3392±0.0062,0.3392±0.0062,18.8671±0.1404 times that in dermis tissue,respectively.The expression in cartilage tissue was 8.0404±0.0635,23.0451±0.9580,0.0489±0.0134,1.6539±0.0422,0.1291±0.0074,6.2856±0.9695 times of that in dermis tissue,respectively.(6)In the middle period,the expression of OPN,PTHLH,STMN2,MDK,MAFK,BTF3L4 gene in mesenchyme tissue was 1.6730±0.2049,0.1036±0.0098,0.3330±0.0886,0.1746±0.0095,0.4594±0.0606,0.4466±0.0446 times that in dermis tissue,respectively.The expression in precartilage was 12.4531±0.9626,1.6761±0.1269,0.5652±0.1083,0.2812±0.0101,2.6262±0.2734,5.2838±0.4685 times of that in dermis tissue,respectively.The expression in cartilage tissue was 14.4736±0.9479,14.0001±0.5287,0.6633±0.2606,1.2679±0.0662,0.1916±0.0049,0.8357±0.0969 times of that in dermis tissue,respectively.(7)In the late period,the expression of OPN,PTHLH,STMN2,MDK,MAFK,BTF3L4 gene in mesenchyme tissue was 1.4229±0.1162,0.5649±0.1640,0.2499±0.0262,0.2256±0.0271,0.4881±0.0499,1.1429±0.0869 times of that in dermis tissue,respectively.The expression in precartilage was 9.0900±0.9705,2.2327±0.6140,2.3976±0.6153,0.6747±0.0636,2.5066±0.0467,5.3638±0.6608 times of that in dermis tissue,respectively.The expression in cartilage tissue was 12.5608±0.9954,2.6762±0.5204,0.4616±0.1698,0.4616±0.1698,0.0859±0.0096,2.0728±0.1581 times of that in dermis tissue,respectively.In conclusion,in this study,OPN,PTHLH,STMN2,MDK,MAFK,BTF3L4 genes were cloned and their cDNA sequences,complete coding sequences and encoded amino acids were obtained for each tissue layer at the tip of antler during its growth process.Further biological information analysis,multiple sequence comparison and real-time quantitative analysis revealed that these genes regulated the growth of antler and were expressed in various tissues at different periods,and there were spatial and temporal differences in expression.These results provided data basis for revealing the internal molecular mechanism of the regulation of the rapid growth of antler,and also provided scientific reference for tissue wound repair and organ regeneration. |
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