鸡lncRNA-MSTRG.15568.9及其预测靶基因的表达
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摘要
试验旨在了解在鸡睾丸中高表达的1个长链非编码RNA(long non-coding RNA,lncRNA)及其预测靶基因的时空表达规律,研究二者在鸡弱精子症中的调控作用。根据弱精子症和正常北京油鸡公鸡睾丸转录组测序筛选到的1个高表达的lncRNA(MSTRG.15568.9),采用顺式(cis)作用模式预测其潜在靶基因SPAG4(sperm-associated antigen 4),进一步采用实时荧光定量PCR方法进行表达量分析。分别选择3只0、5、20、30、45、60周龄正常北京油鸡公鸡,检测MSTRG.15568.9与SPAG4基因在不同周龄公鸡睾丸中的表达量差异;选择30周龄3只正常公鸡,采集睾丸、肝脏和脾脏等8个部位组织样品,检测MSTRG.15568.9与SPAG4基因在不同组织间的表达规律;选择45周龄弱精子症公鸡和正常公鸡各3只,对比MSTRG.15568.9与SPAG4基因在睾丸的表达量差异。结果显示,MSTRG.15568.9与SPAG4存在明显的时空表达差异,且二者表达趋势基本一致。在不同周龄的鸡睾丸组织中,MSTRG.15568.9和SPAG4的表达趋势相近,MSTRG.15568.9在20周龄的表达量显著高于0、5、30、45、60周龄(P<0.05),0和5周龄表达量显著低于20、30、45和60周龄(P<0.05);SPAG4在45周龄表达量最高,其次是20周龄(P<0.05)。MSTRG.15568.9和SPAG4在睾丸和肝脏中的表达量均显著高于脾脏、肾脏等组织(P<0.05);在正常睾丸组织中的表达量均显著高于弱精子症睾丸组织(P<0.05)。综上所述,MSTRG.15568.9与SPAG4基因具有较明显的组织表达特异性,且MSTRG.15568.9可能调控SPAG4基因的表达,参与精子发生与精子活力调控;但其具体作用机制需要进一步探索。本研究可为鉴定与鸡弱精子症调节机制相关的功能基因提供参考。
The objective of this study was to study the spatio-temporal expression of a long non-coding RNA(lncRNA) and its predicted target gene,which are highly expressed in chicken testis,and its regulation in asthenospermia Beijing-You chickens.A high-expression lncRNA(MSTRG.15568.9) was screened by the transcriptome of normal and the asthenospermia Beijing-You chickens.Its potential target gene(sperm-associated antigen 4,SPAG4) was revealed by cis prediction,and RT-qPCR was used to further expression analysis.3 normal cocks with similar body weight were randomly selected at 0,5,20,30,45,and 60 weeks of age to detect the difference in expression between MSTRG.15568.9 and SPAG4 at different ages;At 30 weeks of age,3 normal cocks were selected,and 8 tissues including testis,liver and spleen etc. collected to detect the expression of MSTRG.15568.9 and SPAG4.At 45 weeks of age,3 normal and 3 asthenospermic cocks were selected,and the difference in expression levels between MSTRG.15568.9 and SPAG4 were compared.The results showed that MSTRG.15568.9 and SPAG4 had significant spatio-temporal expression differences,and the expression trends of them were basically the same.The expression trends of MSTRG.15568.9 and SPAG4 were similar in testis tissues of different ages.The expression level of MSTRG.15568.9 at 20 weeks was significantly higher than that at 0,5,30,45 and 60 weeks(P<0.05).The expression levels at 0 and 5 weeks of age were significantly lower than those at 20,30,45 and 60 weeks(P<0.05);SPAG4 was highest at 45 weeks of age,followed by 20 weeks(P<0.05).The expression levels of MSTRG.15568.9 and SPAG4 in testis and liver were significantly higher than that in spleen and kidney(P<0.05).The expression levels of MSTRG.15568.9 and SPAG4 in testis of cocks with normal sperm motility were significantly higher than those in asthenospermia cocks(P<0.05).In conclusion,MSTRG.15568.9 and SPAG4 had obvious tissue expression specificity,and the expression of SPAG4 might be related to MSTRG.15568.9,and both could be involved in spermatogenesis and sperm motility regulation.The specific mechanisms need to be further explored.This study could provide references for the identification of functional genes related to the regulation mechanism of chicken asthenospermia.
The objective of this study was to study the spatio-temporal expression of a long non-coding RNA(lncRNA) and its predicted target gene,which are highly expressed in chicken testis,and its regulation in asthenospermia Beijing-You chickens.A high-expression lncRNA(MSTRG.15568.9) was screened by the transcriptome of normal and the asthenospermia Beijing-You chickens.Its potential target gene(sperm-associated antigen 4,SPAG4) was revealed by cis prediction,and RT-qPCR was used to further expression analysis.3 normal cocks with similar body weight were randomly selected at 0,5,20,30,45,and 60 weeks of age to detect the difference in expression between MSTRG.15568.9 and SPAG4 at different ages;At 30 weeks of age,3 normal cocks were selected,and 8 tissues including testis,liver and spleen etc. collected to detect the expression of MSTRG.15568.9 and SPAG4.At 45 weeks of age,3 normal and 3 asthenospermic cocks were selected,and the difference in expression levels between MSTRG.15568.9 and SPAG4 were compared.The results showed that MSTRG.15568.9 and SPAG4 had significant spatio-temporal expression differences,and the expression trends of them were basically the same.The expression trends of MSTRG.15568.9 and SPAG4 were similar in testis tissues of different ages.The expression level of MSTRG.15568.9 at 20 weeks was significantly higher than that at 0,5,30,45 and 60 weeks(P<0.05).The expression levels at 0 and 5 weeks of age were significantly lower than those at 20,30,45 and 60 weeks(P<0.05);SPAG4 was highest at 45 weeks of age,followed by 20 weeks(P<0.05).The expression levels of MSTRG.15568.9 and SPAG4 in testis and liver were significantly higher than that in spleen and kidney(P<0.05).The expression levels of MSTRG.15568.9 and SPAG4 in testis of cocks with normal sperm motility were significantly higher than those in asthenospermia cocks(P<0.05).In conclusion,MSTRG.15568.9 and SPAG4 had obvious tissue expression specificity,and the expression of SPAG4 might be related to MSTRG.15568.9,and both could be involved in spermatogenesis and sperm motility regulation.The specific mechanisms need to be further explored.This study could provide references for the identification of functional genes related to the regulation mechanism of chicken asthenospermia.
引文
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[5] 许红,孙研研,石雷,等.SOX5蛋白对公鸡精子发生和精子活力的作用及其定位[J].畜牧兽医学报,2018,49(4):718-724.XU H,SUN Y Y,SHI L,et al.Effects and localization of SOX5 protein on spermatogenesis and sperm motility in cock[J].Chinese Journal of Animal and Veterinary Sciences,2018,49(4):718-724.(in Chinese)
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[8] LIU Y,SUN Y,LI Y,et al.Identification and differential expression of microRNAs in the testis of chicken with high and low sperm motility[J].Theriogenology,2018,122:94-101.
[9] WU Q,SONG R,ORTOGERO N,et al.The RNase Ⅲ enzyme Drosha is essential for microRNA production and spermatogenesis[J].Journal of Biological Chemistry,2012,287(30):25173.
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[11] 苏晓娜,谢青梅,陈峰.长链非编码RNA及其在畜禽生长调控中的研究进展[J].中国畜牧兽医,2016,43(1):197-203.SU X N,XIE Q M,CHEN F.Research progress of long non-coding RNA and its regulation in livestock and poultry growth[J].China Animal Husbandry & Veterinary Medicine,2016,43(1):197-203.(in Chinese)
[12] FU L L,XU Y,LI D D,et al.Expression profiles of mRNA and long non-coding RNA in the ovaries of letrozole-induced polycystic ovary syndrome rat model through deep sequencing[J].Gene,2018,8(60):101649-101658.
[13] HAN D X,SUN X L,FU Y,et al.Identification of long non-coding RNAs in the immature and mature rat anterior pituitary[J].Scientific Reports,2017,7(1):17780.
[14] LIU Y,SUN Y,LI Y,et al.Analyses of long non-coding RNA and mRNA profiling using RNA sequencing in chicken testis with extreme sperm motility[J].Scientific Reports,2017,7(1):9055.
[15] LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method[J].Methods,2001,25(4):402-408.
[16] OKAZAKI Y,FURUNO M,KASUKAWA T,et al.Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs[J].Nature,2002,420(6915):563-573.
[17] HANGAUER M J,VAUGHN I W,MCMANUS M T.Pervasive transcription of the human genome produces thousands of previously unidentified long intergenic noncoding RNAs[J].PLoS Genetics,2013,9(6):e1003569.
[18] MERCER T R,DINGER M E,MATTICK J S.Long non-coding RNAs:Insights into functions[J].Nature Reviews Genetics,2009,10(3):155-159.
[19] TWAYANA S,LEGNINI I,CESANA M,et al.Biogenesis and function of non-coding RNAs in muscle differentiation and in Duchenne muscular dystrophy[J].Biochemical Society Transactions,2013,41(4):844-849.
[20] LAIHO A,KOTAJA N,GYENESEI A,et al.Transcriptome profiling of the murine testis during the first wave of spermatogenesis[J].PLoS One,2013,8(4):e61558.
[21] 韩聪.山羊生精细胞发育及睾丸组织lncRNA差异表达分析[D].杨凌:西北农林科技大学,2017.HAN C.Differential expression analysis of spermatogenic cell development and testicular tissue lncRNA in goats [D].Yangling:Northwest A & F University,2017.(in Chinese)
[22] 杨花.性成熟前后湖羊睾丸差异lncRNA表达谱分析及关键lncRNA TCONS_00863147功能初步验证[A].2018年全国养羊生产与学术研讨会论文集[C].2018.YANG H.Analysis of lncRNA expression profile of lake sheep testis before and after sexual maturity and preliminary verification of key lncRNA TCONS_00863147 function[A].2018 National Sheep Production and Symposium Proceedings[C].2018.(in Chinese)
[23] SOUMILLON M,NECSULEA A,WEIER M,et al.Cellular source and mechanisms of high transcriptome complexity in the mammalian testis[J].Cell Reports,2013,3(6):2179-2190.
[24] ANGUERA M C,MA W,CLIFT D,et al.Tsx produces a long noncoding RNA and has general functions in the germline,stem cells,and brain[J].PLoS Genetics,2011,7(9):15-24.
[25] ZHENG G,DAHL J A,NIU Y,et al.ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility[J].Molecular Cell,2013,49(6):18-29.
[26] RAN M,CHEN B,LI Z,et al.Systematic identification of long noncoding RNAs in immature and mature porcine testes[J].Biology of Reproduction,2016,94(4):77.
[27] ZHANG Y,YANG H,HAN L,et al.Long noncoding RNA expression profile changes associated with dietary energy in the sheep testis during sexual maturation[J].Scientific Reports,2017,7(1):5180.
[28] TRAPNELL C,WILLIAMS B A,et al.Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation[J].Nature Biotechnology,2010,28(5):511-515.
[29] KOUFARIOTIS L T,PHOEBE Y P,AMANDA C,et al.A catalogue of novel bovine long noncoding RNA across 18 tissues[J].PLoS One,2015,10(10):e0141225.
[30] KERR L E,PATERSON M,AITKEN R J.Molecular basis of sperm-egg interaction and the prospects for immunocontraception[J].Journal of Reproductive Immunology,1998,40(2):103.
[31] 许红.SPAG6和SOX5蛋白对公鸡精子发生和精子活力作用的研究[D].北京:中国农业科学院,2017.XU H.Study on the effects of SPAG6 and SOX5 proteins on spermatogenesis and sperm motility in cocks[D].Beijing:Chinese Academy of Agricultural Sciences,2017.(in Chinese)
[32] SHAO X,FA V D H.Self-interaction of the major 27-kilodalton outer dense fiber protein is in part mediated by a leucine zipper domain in the rat[J].Biology of Reproduction,1996,55(6):1343-1350.
[33] KENNEDY C,SEBIREK,KRETSER D,et al.Human sperm associated antigen 4 (SPAG4) is a potential cancer marker[J].Cell & Tissue Research,2004,315(2):279-283.
[34] PASCH E,LINK J,BECK C,et al.The LINC complex component Sun4 plays a crucial role in sperm head formation and fertility[J].Biology Open,2015,4(12):1792-1802.
[35] CALVI A,WONG A S,WRIGHT G,et al.SUN4 is essential for nuclear remodeling during mammalian spermiogenesis[J].Developmental Biology,2015,407(2):321-330.
[36] SHAO X,TARNASKY H A,LEE J P,et al.Spag4,a novel sperm protein,binds outer dense-fiber protein Odf1 and localizes to microtubules of manchette and axoneme[J].Developmental Biology,1999,211(1):109-123.
[37] JI Y,JIANG J,HUANG L,et al.Sperm associated antigen 4 (SPAG4) as a new cancer marker interacts with Nesprin3 to regulate cell migration in lung carcinoma[J].Oncology Reports,2018,40(2):1-10.
[2] GUNAWAN A,CINAR M U,UDDIN M J,et al.Investigation on association and expression of ESR2 as a candidate gene for boar sperm quality and fertility[J].Domestic Animals,2012,47(5):782-790.
[3] KAEWMALA K,UDDIN M J,CINAR M U,et al.Association study and expression analysis of CD9 as candidate gene for boar sperm quality and fertility traits[J].Animal Reproduction Science,2011,125(1-4):170-179.
[4] SASAKI Y,KOHSAKA T,KAWARASAKI T,et al.Immunoreactive relaxin in seminal plasma of fertile boars and its correlation with sperm motility characteristics determined by computer-assisted digital image analysis[J].International Journal of Andrology,2001,24(1):24-30.
[5] 许红,孙研研,石雷,等.SOX5蛋白对公鸡精子发生和精子活力的作用及其定位[J].畜牧兽医学报,2018,49(4):718-724.XU H,SUN Y Y,SHI L,et al.Effects and localization of SOX5 protein on spermatogenesis and sperm motility in cock[J].Chinese Journal of Animal and Veterinary Sciences,2018,49(4):718-724.(in Chinese)
[6] YAN N,LU Y,SUN H,et al.A microarray for microRNA profiling in mouse testis tissues[J].Reproduction,2007,134(1):73-79.
[7] GAN H,LIN X,ZHANG Z,et al.piRNA profiling during specific stages of mouse spermatogenesis[J].RNA,2011,17(7):1191-1203.
[8] LIU Y,SUN Y,LI Y,et al.Identification and differential expression of microRNAs in the testis of chicken with high and low sperm motility[J].Theriogenology,2018,122:94-101.
[9] WU Q,SONG R,ORTOGERO N,et al.The RNase Ⅲ enzyme Drosha is essential for microRNA production and spermatogenesis[J].Journal of Biological Chemistry,2012,287(30):25173.
[10] 陆启荣,刘孟轲,李丽,等.畜禽长链非编码RNA的功能及其研究方法[J].中国畜牧兽医,2016,43(9):2425-2434.LU Q R,LIU M K,LI L,et al.The function and research method of long non-coding RNA in livestock and poultry[J].China Animal Husbandry & Veterinary Medicine,2016,43(9):2425-2434.(in Chinese)
[11] 苏晓娜,谢青梅,陈峰.长链非编码RNA及其在畜禽生长调控中的研究进展[J].中国畜牧兽医,2016,43(1):197-203.SU X N,XIE Q M,CHEN F.Research progress of long non-coding RNA and its regulation in livestock and poultry growth[J].China Animal Husbandry & Veterinary Medicine,2016,43(1):197-203.(in Chinese)
[12] FU L L,XU Y,LI D D,et al.Expression profiles of mRNA and long non-coding RNA in the ovaries of letrozole-induced polycystic ovary syndrome rat model through deep sequencing[J].Gene,2018,8(60):101649-101658.
[13] HAN D X,SUN X L,FU Y,et al.Identification of long non-coding RNAs in the immature and mature rat anterior pituitary[J].Scientific Reports,2017,7(1):17780.
[14] LIU Y,SUN Y,LI Y,et al.Analyses of long non-coding RNA and mRNA profiling using RNA sequencing in chicken testis with extreme sperm motility[J].Scientific Reports,2017,7(1):9055.
[15] LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method[J].Methods,2001,25(4):402-408.
[16] OKAZAKI Y,FURUNO M,KASUKAWA T,et al.Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs[J].Nature,2002,420(6915):563-573.
[17] HANGAUER M J,VAUGHN I W,MCMANUS M T.Pervasive transcription of the human genome produces thousands of previously unidentified long intergenic noncoding RNAs[J].PLoS Genetics,2013,9(6):e1003569.
[18] MERCER T R,DINGER M E,MATTICK J S.Long non-coding RNAs:Insights into functions[J].Nature Reviews Genetics,2009,10(3):155-159.
[19] TWAYANA S,LEGNINI I,CESANA M,et al.Biogenesis and function of non-coding RNAs in muscle differentiation and in Duchenne muscular dystrophy[J].Biochemical Society Transactions,2013,41(4):844-849.
[20] LAIHO A,KOTAJA N,GYENESEI A,et al.Transcriptome profiling of the murine testis during the first wave of spermatogenesis[J].PLoS One,2013,8(4):e61558.
[21] 韩聪.山羊生精细胞发育及睾丸组织lncRNA差异表达分析[D].杨凌:西北农林科技大学,2017.HAN C.Differential expression analysis of spermatogenic cell development and testicular tissue lncRNA in goats [D].Yangling:Northwest A & F University,2017.(in Chinese)
[22] 杨花.性成熟前后湖羊睾丸差异lncRNA表达谱分析及关键lncRNA TCONS_00863147功能初步验证[A].2018年全国养羊生产与学术研讨会论文集[C].2018.YANG H.Analysis of lncRNA expression profile of lake sheep testis before and after sexual maturity and preliminary verification of key lncRNA TCONS_00863147 function[A].2018 National Sheep Production and Symposium Proceedings[C].2018.(in Chinese)
[23] SOUMILLON M,NECSULEA A,WEIER M,et al.Cellular source and mechanisms of high transcriptome complexity in the mammalian testis[J].Cell Reports,2013,3(6):2179-2190.
[24] ANGUERA M C,MA W,CLIFT D,et al.Tsx produces a long noncoding RNA and has general functions in the germline,stem cells,and brain[J].PLoS Genetics,2011,7(9):15-24.
[25] ZHENG G,DAHL J A,NIU Y,et al.ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility[J].Molecular Cell,2013,49(6):18-29.
[26] RAN M,CHEN B,LI Z,et al.Systematic identification of long noncoding RNAs in immature and mature porcine testes[J].Biology of Reproduction,2016,94(4):77.
[27] ZHANG Y,YANG H,HAN L,et al.Long noncoding RNA expression profile changes associated with dietary energy in the sheep testis during sexual maturation[J].Scientific Reports,2017,7(1):5180.
[28] TRAPNELL C,WILLIAMS B A,et al.Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation[J].Nature Biotechnology,2010,28(5):511-515.
[29] KOUFARIOTIS L T,PHOEBE Y P,AMANDA C,et al.A catalogue of novel bovine long noncoding RNA across 18 tissues[J].PLoS One,2015,10(10):e0141225.
[30] KERR L E,PATERSON M,AITKEN R J.Molecular basis of sperm-egg interaction and the prospects for immunocontraception[J].Journal of Reproductive Immunology,1998,40(2):103.
[31] 许红.SPAG6和SOX5蛋白对公鸡精子发生和精子活力作用的研究[D].北京:中国农业科学院,2017.XU H.Study on the effects of SPAG6 and SOX5 proteins on spermatogenesis and sperm motility in cocks[D].Beijing:Chinese Academy of Agricultural Sciences,2017.(in Chinese)
[32] SHAO X,FA V D H.Self-interaction of the major 27-kilodalton outer dense fiber protein is in part mediated by a leucine zipper domain in the rat[J].Biology of Reproduction,1996,55(6):1343-1350.
[33] KENNEDY C,SEBIREK,KRETSER D,et al.Human sperm associated antigen 4 (SPAG4) is a potential cancer marker[J].Cell & Tissue Research,2004,315(2):279-283.
[34] PASCH E,LINK J,BECK C,et al.The LINC complex component Sun4 plays a crucial role in sperm head formation and fertility[J].Biology Open,2015,4(12):1792-1802.
[35] CALVI A,WONG A S,WRIGHT G,et al.SUN4 is essential for nuclear remodeling during mammalian spermiogenesis[J].Developmental Biology,2015,407(2):321-330.
[36] SHAO X,TARNASKY H A,LEE J P,et al.Spag4,a novel sperm protein,binds outer dense-fiber protein Odf1 and localizes to microtubules of manchette and axoneme[J].Developmental Biology,1999,211(1):109-123.
[37] JI Y,JIANG J,HUANG L,et al.Sperm associated antigen 4 (SPAG4) as a new cancer marker interacts with Nesprin3 to regulate cell migration in lung carcinoma[J].Oncology Reports,2018,40(2):1-10.