滩羊Eph A3基因克隆表达分析及生物信息学初步研究
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摘要
本研究以滩羊不同时期(1月龄和48月龄)被毛卷曲性状差异为基础,对在前期构建的消减文库中获得与滩羊被毛卷曲形成相关的重要候选基因EphA3进一步分析。首先通过PCR扩增技术获得EphA3基因的编码区CDS全长,并利用生物信息学方法分析该基因所编码的蛋白结构及其在不同物种中的同源性和进化关系。结果表明:滩羊EphA3基因序列在不同物种间高度保守,在皮肤组织中表达量最高;该基因在48月龄滩羊皮肤组织中的表达量显著高于1月龄个体(P<0.05)。EphA3基因可能是影响不同时期滩羊被毛卷曲差异的候选基因之一。
This study aimed to elucidate the structure,physiological function and the expression levels of EphA3 gene at different stage(one-month-old and 48-month-old)in Tan sheep.The EphA3 gene in Tan sheep was amplified through PCR using extracted RNA from skin samples.Then the gene was sequenced and analyzed by bioinformatic tools.Bioinformatic tools were used to analyze the CDS region and the secondary structure of the EphA3 gene.Phylogenetic tree and sequence alignment showed the nucleotide sequence of EphA3 gene has a high homology between Chinese Tan sheep and other species,which indicates EphA3 was relatively conserved.Semi-quantitative RT-PCR analysis showed that EphA3 gene was higher expressed in skin than other tissues.Further q-PCR data from skin showed that the expression of EphA3 gene at 48-month-old was significantly higher than that at one-month-old(P<0.05).All the results suggest that EphA3 gene may play an important role in curly fleece formation mechanism in Chinese Tan sheep,and provide some valuable clues for further studies on biology characterization of curly fleece growth.
This study aimed to elucidate the structure,physiological function and the expression levels of EphA3 gene at different stage(one-month-old and 48-month-old)in Tan sheep.The EphA3 gene in Tan sheep was amplified through PCR using extracted RNA from skin samples.Then the gene was sequenced and analyzed by bioinformatic tools.Bioinformatic tools were used to analyze the CDS region and the secondary structure of the EphA3 gene.Phylogenetic tree and sequence alignment showed the nucleotide sequence of EphA3 gene has a high homology between Chinese Tan sheep and other species,which indicates EphA3 was relatively conserved.Semi-quantitative RT-PCR analysis showed that EphA3 gene was higher expressed in skin than other tissues.Further q-PCR data from skin showed that the expression of EphA3 gene at 48-month-old was significantly higher than that at one-month-old(P<0.05).All the results suggest that EphA3 gene may play an important role in curly fleece formation mechanism in Chinese Tan sheep,and provide some valuable clues for further studies on biology characterization of curly fleece growth.
引文
[1]Genander M, Holmberg J, Frisén J. Ephrins negatively regulatecell proliferation in the epidermis and hair follicle[J]. Stem Cells,2010, 28(7):1196-1205.
[2]Diao X, Chen X, Pi Y, et al. Androgen receptor inducesEPHA3 expression by interacting with transcription factorSP1[J]. Oncol Rep, 2018, 40(2):1174-1184.
[3]Nishikimi M, Oishi K, Tabata H, et al. Seg regation andpathfinding of callosal axons through EphA3 signaling[J]. JNeurosci, 2011, 31(45):16251-16260.
[4]Higenell V, Han S M, Feldheim D A, et al. Expression patternsof Ephs and ephrins throughout retinotectal development inXenopus laevis[J]. Dev Neurobiol, 2012, 72(4):547-563.
[5]Rocca F L, Airoldi I, Carlo E D, et al. Eph A3 targetingreduces in vitro adhesion and invasion and in vivo growth andangiogenesis of multiple myeloma cells[J]. Cell Oncol, 2017,40(Suppl 1):1-14.
[6]Yamada Y, Midorikawa T, Oura H, et al. Ephrin-A3 not onlyincreases the density of hair follicles but also acceleratesanagen development in neonatal mice[J]. J Dermatol Sci,2008, 52(3):178-185.
[7]Zouboulis C C, Adjaye J, Akamatsu H, et al. Human skin stemcells and the ageing process[J]. Exp Gerontol, 2008, 43(11):986-997.
[8]Tumbar T, Guasch G, Greco V, et al. Defining the epithelialstem cell niche in skin[J]. Sci Signal, 2004, 303(5656):359.
[9]Kang X, Liu G, Liu Y, et al. Transcriptome profile at differentphysiological stages reveals potential mode for curly fleece inchinese tan sheep[J]. PLoS One, 2013, 8(8):e71763.
[10]崔重九,张幼麟,蒋英,等.滩羊选育报告(第二报)——关于滩羊二毛皮品质的研究[J].中国畜牧兽医, 1962(7):1-3.
[11]白玲荣,杨佐青,陶金忠.滩羊羊毛蛋白质双向电泳图谱体系的建立及优化[J].中国草食动物科学, 2018(1):18-22.
[2]Diao X, Chen X, Pi Y, et al. Androgen receptor inducesEPHA3 expression by interacting with transcription factorSP1[J]. Oncol Rep, 2018, 40(2):1174-1184.
[3]Nishikimi M, Oishi K, Tabata H, et al. Seg regation andpathfinding of callosal axons through EphA3 signaling[J]. JNeurosci, 2011, 31(45):16251-16260.
[4]Higenell V, Han S M, Feldheim D A, et al. Expression patternsof Ephs and ephrins throughout retinotectal development inXenopus laevis[J]. Dev Neurobiol, 2012, 72(4):547-563.
[5]Rocca F L, Airoldi I, Carlo E D, et al. Eph A3 targetingreduces in vitro adhesion and invasion and in vivo growth andangiogenesis of multiple myeloma cells[J]. Cell Oncol, 2017,40(Suppl 1):1-14.
[6]Yamada Y, Midorikawa T, Oura H, et al. Ephrin-A3 not onlyincreases the density of hair follicles but also acceleratesanagen development in neonatal mice[J]. J Dermatol Sci,2008, 52(3):178-185.
[7]Zouboulis C C, Adjaye J, Akamatsu H, et al. Human skin stemcells and the ageing process[J]. Exp Gerontol, 2008, 43(11):986-997.
[8]Tumbar T, Guasch G, Greco V, et al. Defining the epithelialstem cell niche in skin[J]. Sci Signal, 2004, 303(5656):359.
[9]Kang X, Liu G, Liu Y, et al. Transcriptome profile at differentphysiological stages reveals potential mode for curly fleece inchinese tan sheep[J]. PLoS One, 2013, 8(8):e71763.
[10]崔重九,张幼麟,蒋英,等.滩羊选育报告(第二报)——关于滩羊二毛皮品质的研究[J].中国畜牧兽医, 1962(7):1-3.
[11]白玲荣,杨佐青,陶金忠.滩羊羊毛蛋白质双向电泳图谱体系的建立及优化[J].中国草食动物科学, 2018(1):18-22.