猪lncRNA-ENSSSCT00000018610的克隆及其在猪卵泡中的表达
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摘要
为了探究lncRNA-ENSSSCT00000018610在猪卵泡发育过程中的作用,本研究采用RACE技术对lncRNA-ENSSSCT00000018610的全长序列进行了扩增克隆,用Real-time PCR进行组织表达谱分析,并分析其在梅山猪和杜洛克猪卵泡中的表达情况以及采用顺式和反式两种方法对lncRNA-ENSSSCT00000018610靶基因进行预测。结果表明,克隆获得了lncRNA-ENSSSCT00000018610全长序列1 731bp;lncRNA-ENSSSCT00000018610在肌肉、心、肝、脾、肺、肾、十二指肠、卵巢、输卵管、子宫、垂体、黄体、下丘脑中均有不同程度的表达,其中以肾、肺、子宫、卵巢表达量相对较高;lncRNA-ENSSSCT00000018610在杜洛克猪S、L卵泡中的表达量显著高于梅山猪(P<0.05),在杜洛克猪M1和M2卵泡中的表达水平极显著高于梅山猪(P<0.01)。lncRNA-ENSSSCT00000018610的潜在靶基因TNIP1、CYP2J2、SCARB1、IBSP等与猪产仔性状相关,并且参与猪的卵泡发育。提示其可能通过对靶基因的调控间接参与卵泡发育过程。
To explore the role of lncRNA-ENSSSCT00000018610 in the development of porcine follicles,the full length RNA sequence of lncRNA-ENSSSCT00000018610 was amplified by RACE technique,it's tissue expression profile and expression pattern in ovarian follicles of Meishan and Duroc sows were analyzed by Real-time PCR,and it's target genes were predicted by using the cis and trans methods.A 1 731 bp-length sequence was obtained as the full length RNA sequence of lncRNA-ENSSSCT00000018610.lncRNA-ENSSSCT00000018610 was expressed differentially in variety of tissues(muscle,heart,liver,spleen,lung,kidney,duodenum,ovary,fallopian tube,uterus,pituitary,corpus luteum,hypothalamus),and expressed relatively higher in kidney,lung,uterus and ovary.The expression level of lncRNA-ENSSSCT00000018610 in S and L follicles of Duroc were significantly higher than that of Meishan(P<0.05).The expression level of lncRNA-ENSSSCT00000018610 in M1 and M2 follicles of Duroc were significantly higher than that of Meishan(P<0.01). The potential target genes of lncRNA-ENSSSCT00000018610 including TNIP1,CYP2 J2,SCARB1 and IBSP were related to the reproduc-tion traits and participated in follicular development of pig.This result suggests that lncRNAENSSSCT00000018610 may participate in follicular development indirectly by regulating its target genes expression.
To explore the role of lncRNA-ENSSSCT00000018610 in the development of porcine follicles,the full length RNA sequence of lncRNA-ENSSSCT00000018610 was amplified by RACE technique,it's tissue expression profile and expression pattern in ovarian follicles of Meishan and Duroc sows were analyzed by Real-time PCR,and it's target genes were predicted by using the cis and trans methods.A 1 731 bp-length sequence was obtained as the full length RNA sequence of lncRNA-ENSSSCT00000018610.lncRNA-ENSSSCT00000018610 was expressed differentially in variety of tissues(muscle,heart,liver,spleen,lung,kidney,duodenum,ovary,fallopian tube,uterus,pituitary,corpus luteum,hypothalamus),and expressed relatively higher in kidney,lung,uterus and ovary.The expression level of lncRNA-ENSSSCT00000018610 in S and L follicles of Duroc were significantly higher than that of Meishan(P<0.05).The expression level of lncRNA-ENSSSCT00000018610 in M1 and M2 follicles of Duroc were significantly higher than that of Meishan(P<0.01). The potential target genes of lncRNA-ENSSSCT00000018610 including TNIP1,CYP2 J2,SCARB1 and IBSP were related to the reproduc-tion traits and participated in follicular development of pig.This result suggests that lncRNAENSSSCT00000018610 may participate in follicular development indirectly by regulating its target genes expression.
引文
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[33]SHI F,LAPOLT P S.Relationship between FoxO1protein levels and follicular development,atresia,and luteinization in the rat ovary[J].J Endocrinol,2003,179(2):195-203.
[34]丁威.二花脸猪卵泡形成和早期发育的特性及其相关通路的研究[D].南京:南京农业大学,2010.DING W.Study on characteristics and signaling pathways involved in follicular formation and early development of erhualian pigs[D].Nanjing:Nanjing Agricultural University,2010.(in Chinese)
[2]YANG L,DUFF M O,GRAVELEY B R,et al.Genomewide characterization of non-polyadenylated RNAs[J].Genome Biol,2011,12(2):R16.
[3]PONTING C P,OLIVER P L,REIK W.Evolution and functions of long noncoding RNAs[J].Cell,2009,136(4):629-641.
[4]VERGARA I A,ERHO N,TRICHE T J,et al.Genomic“Dark Matter”in prostate cancer:exploring the clinical utility of ncRNA as biomarkers[J].Front Genet,2012,3:23.
[5]吴昊,张运海,凌英会.非编码RNA在胚胎发育过程中的作用[J].畜牧兽医学报,2017,48(1):1-7.WU H,ZHANG Y H,LING H Y.The progress of recent advances in the ncRNA-mediated regulation during embryogenesis[J].Acta Veterinaria et Zootechnica Sinica,2017,48(1):1-7.(in Chinese)
[6]NAKAYA H I,AMARAL P P,LOURO R,et al.Genome mapping and expression analyses of human intronic noncoding RNAs reveal tissue-specific patterns and enrichment in genes related to regulation of transcription[J].Genome Biol,2007,8(3):R43.
[7]STRUHL K.Transcriptional noise and the fidelity of initiation by RNA polymerase II[J].Nat Struct Mol Biol,2007,14(2):103-105.
[8]DERRIEN T,JOHNSON R,BUSSOTTI G,et al.The GENCODE v7 catalog of human long noncoding RNAs:analysis of their gene structure,evolution,and expression[J].Genome Res,2012,22(9):1775-1789.
[9]杨峰,易凡,曹慧青,等.长链非编码RNA研究进展[J].遗传,2014,36(5):456-468.YANG F,YI F,CAO H Q,et al.The emerging landscape of long non-coding RNAs[J].Hereditas,2014,36(5):456-468.(in Chinese)
[10]YAN L Y,YANG M Y,GUO H S,et al.Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells[J].Nat Struct Mol Biol,2013,20(9):1131-1139.
[11]HAMAZAKI N,UESAKA M,NAKASHIMA K,et al.Gene activation-associated long noncoding RNAs function in mouse preimplantation development[J].Development,2015,142(5):910-920.
[12]CABALLERO J,GILBERT I,FOURNIER E,et al.Exploring the function of long non-coding RNA in the development of bovine early embryos[J].Reprod Fertil Dev,2015,27(1):40-52.
[13]KANEKO S,SON J,SHEN S S,et al.PRC2binds active promoters and contacts nascent RNAs in embryonic stem cells[J].Nat Struct Mol Biol,2013,20(11):1258-1264.
[14]CHEN H J,PALMER J S,THIAGARAJAN R D,et al.Identification of novel markers of mouse fetal ovary development[J].PLoS One,2012,7(7):e41683.
[15]ROESZLER K N,ITMAN C,SINCLAIR A H,et al.The long non-coding RNA,MHM,plays a role in chicken embryonic development,including gonadogenesis[J].Dev Biol,2012,366(2):317-326.
[16]BROWN J B,BOLEY N,EISMAN R,et al.Diversity and dynamics of the Drosophila transcriptome[J].Nature,2014,512(7515):393-399.
[17]MACAULAY A D,GILBERT I,CABALLERO J,et al.The gametic synapse:RNA transfer to the bovine oocyte[J].Biol Reprod,2014,91(4):90.
[18]马力鹏.杜洛克猪与梅山猪中等猪卵泡差异表达lncRNA和mRNA的鉴定研究[D].石河子:河子大学,2017.MA L P.Identification of differentially expressed lncRNA and mRNA between medium follicles in Duroc and Meishan Pig[D].Shihezi:Shihezi University,2017.(in Chinese)
[19]WANG L G,PARK H J,DASARI S,et al.CPAT:coding-potential assessment tool using an alignmentfree logistic regression model[J].Nucleic Acids Res,2013,41(6):e74.
[20]ZHOU Z Y,LI A M,ADEOLA A C,et al.Genomewide identification of long intergenic noncoding RNA genes and their potential association with domestication in pigs[J].Genome Biol Evol,2014,6(6):1387-1392.
[21]HUNG T,CHANG H Y.Long noncoding RNA in genome regulation:prospects and mechanisms[J].RNA Biol,2010,7(5):582-585.
[22]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the method[J].Methods,2001,25(4):402-408.
[23]林海军.胚胎中lncRNA的预测及其在胚胎发育过程中的功能分析[D].南昌:南昌大学,2015.LIN H J.The prediction and functional analysis of lncRNA in embryonic development process[D].Nanchang:Nanchang University,2015.(in Chinese)
[24]WANG Y Y,XUE S Y,LIU X R,et al.Analyses of long non-coding RNA and mRNA profiling using RNA sequencing during the pre-implantation phases in pig endometrium[J].Sci Rep,2016,6:20238.
[25]LIANU M,LI W Q,TIAN H,et al.Sequential expression of long noncoding RNA as mRNA gene expression in specific stages of mouse spermatogenesis[J].Sci Rep,2014,4:5966.
[26]WASHIETL S,HOFACKER I L,STADLER P F.Fast and reliable prediction of noncoding RNAs[J].Proc Natl Acad Sci U S A,2015,102(7):2454-2459.
[27]王羽.基于序列-结构信息的长非编码RNA预测方法[D].西安:西安电子科技大学,2014.WANG Y.Method for lncRNA prediction based on sequence-structure information[D].Xi’an:Xidian University,2014.(in Chinese)
[28]贾纯琰,季小阳,白雪,等.长链非编码RNA的调控机制及其在家畜中的预测方法[J].中国畜牧兽医,2017,44(7):1895-1905.JIA C Y,JI X Y,BAI X,et al.Regulatory mechanisms of long noncoding RNAs and forecasting methods in livestock[J].China Animal Husbandry&Veterinary Medicine,2017,44(7):1895-1905.(in Chinese)
[29]DINGER M E,PAND K C,MERCER T R,et al.Differentiating protein-coding and noncoding RNA:challenges and ambiguities[J].PLoS Comput Biol,2008,4(11):e1000176.
[30]PAULI A,VALEN E,SCHIER A F,et al.Identifying(non-)coding RNAs and small peptides:challenges and opportunities[J].Bioessays,2015,37(1):103-112.
[31]周晓宁,何小梅,贾新正,等.ABIN-1基因5个SNP对长白母猪产仔数的影响[C]//第十二次全国畜禽遗传标记研讨会.南京:中国畜牧兽医学会,2010.ZHOU X N,HE X M,JIA X Z,et al.Effect of 5SNPs of ABIN-1gene on litter size in Landrace Sows[C]//Twelfth National Symposium on Livestock and Poultry Genetic Markers.Nanjing:Chinese Animal Husbandry and Veterinary Association,2010.(in Chinese)
[32]欧阳宏佳,方梅霞,周晓宁,等.猪胚胎发育相关基因ABIN-1的cDNA克隆及表达[C]//中国动物遗传育种研究进展——第十五次全国动物遗传育种学术讨论会.杨凌:中国畜牧兽医学会,2009.OUYANG H J,FANG M X,ZHOU X N,et al.cDNA cloning and expression of porcine embryonic development related gene ABIN-1[C]//Progress in Animal Genetics and Breeding in China:the 15th National Conference on Animal Genetics and Breeding Collection.Yangling:Chinese Animal Husbandry and Veterinary Association,2009.(in Chinese)
[33]SHI F,LAPOLT P S.Relationship between FoxO1protein levels and follicular development,atresia,and luteinization in the rat ovary[J].J Endocrinol,2003,179(2):195-203.
[34]丁威.二花脸猪卵泡形成和早期发育的特性及其相关通路的研究[D].南京:南京农业大学,2010.DING W.Study on characteristics and signaling pathways involved in follicular formation and early development of erhualian pigs[D].Nanjing:Nanjing Agricultural University,2010.(in Chinese)