版纳微型猪近交系雄激素受体AR基因的克隆和表达分析
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摘要
为探讨猪雄激素受体(Androgen receptor,AR)基因的序列、结构、表达和功能,以版纳微型猪近交系(Banna mini-pig inbred line,BMI)睾丸组织为材料,用RT-PCR技术获得猪AR基因cDNA序列,进行生物信息学分析,应用荧光定量技术明确其mRNA的多组织表达特征。结果表明:BMI AR基因cDNA长3 257bp(GenBank登录号:KU705631),包含2 688bp的全长编码区,位于猪X染色体,含有9个外显子和8个内含子;推导出的BMI AR蛋白含有1个核定位信号、1个DBD结构域和1个LBD结构域,无信号肽,无跨膜螺旋;与黄牛、山羊、人、猩猩、大鼠和小鼠的AR蛋白依次有94.2%、94.0%、90.2%、89.0%、88.1%和87.5%的同源性;其二级结构中以无规则卷曲和α螺旋为主,延伸链和β转角只在局部出现;在二级结构预测结果的基础上利用同源建模构建了其三级结构;AR基因mRNA在被检测的BMI 15个组织中呈现差异表达现象,表达量最高的组织为睾丸。该研究结果为进一步研究AR基因在BMI中的生理功能及调节机制提供了依据。
In order to explore sequence and structure of pig androgen receptor(AR),the cDNA of ARgene was cloned from the testis of Banna mini-pig inbred line(BMI)by RT-PCR for sequencing and characterization of AR was analyzed by bioinformatics analysis.The expression pattern of ARin different tissues was detected by qPCR.A cDNA of 3 257 bp(GenBank accession number:KU705631)of BMI AR was obtained,which contained 2 688 bp opening reading frame.Genome structure analysis showed it localized to Sus scrofa chromosomes X and was consisted of nine exons and eight introns.Functional bioinformatics analysis indicated that BMI AR protein contained one nuclear location signal,one DBD domain,one LBD domain,no transmembrane region and no signal peptide.The predicted amino acid sequence of this gene had high homology with the cattle(94.2% ),goat(94.0% ),human(90.2% ),chimpanzee(89.0% ),rat(88.1% )and mouse(87.5% ).The secondary structure of BMI AR protein was mainly consisted of random coils andα-helices,βturns and extended strands were locally present.Its tertiary structure was also predicted by homology modeling based on the secondary structure data.AR mRNA was differentially expressed in the 15 tissues of BMI with the highest expression level in testis.This research provided foundations for further study on the physiological function and regulatory mechanisms of AR gene in BMI.
In order to explore sequence and structure of pig androgen receptor(AR),the cDNA of ARgene was cloned from the testis of Banna mini-pig inbred line(BMI)by RT-PCR for sequencing and characterization of AR was analyzed by bioinformatics analysis.The expression pattern of ARin different tissues was detected by qPCR.A cDNA of 3 257 bp(GenBank accession number:KU705631)of BMI AR was obtained,which contained 2 688 bp opening reading frame.Genome structure analysis showed it localized to Sus scrofa chromosomes X and was consisted of nine exons and eight introns.Functional bioinformatics analysis indicated that BMI AR protein contained one nuclear location signal,one DBD domain,one LBD domain,no transmembrane region and no signal peptide.The predicted amino acid sequence of this gene had high homology with the cattle(94.2% ),goat(94.0% ),human(90.2% ),chimpanzee(89.0% ),rat(88.1% )and mouse(87.5% ).The secondary structure of BMI AR protein was mainly consisted of random coils andα-helices,βturns and extended strands were locally present.Its tertiary structure was also predicted by homology modeling based on the secondary structure data.AR mRNA was differentially expressed in the 15 tissues of BMI with the highest expression level in testis.This research provided foundations for further study on the physiological function and regulatory mechanisms of AR gene in BMI.
引文
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[15]陈园园,潘伟荣,霍金龙,王淑燕,王配,查星琴,曾养志.版纳微型猪近交系不育和可育公猪CRISP2基因全长编码区序列克隆及睾丸差异表达分析[J].云南农业大学学报自然科学,2015,30(5):698-705Chen Y Y,Pan W R,Huo J L,Wang S Y,Wang P,Zha X Q,Zeng Y Z.Cloning and testis expression difference analysis of coding region sequence of CRISP2gene in sterile and fertile boar of Banna mini-pig inbred line[J].Journal of Yunnan Agricultural University:Natural Science,2015,30(5):698-705(in Chinese)
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[18]Pu L,Han K,Xie F,Zou Z,Close D,Zhang Z,Wang Y.Molecular cloning,characterization,and gene expression of the androgen receptor in the large yellow croaker,Larimichthys crocea[J].Fish Physiology and Biochemistry,2013,39(2):309-324
[19]Simental J A,Sar M,Lane M V,French F S,Wilson E M.Transcriptional activation and nuclear targeting signals of the human androgen receptor[J].Journal of Biological Chemistry,1991,266(1):510-518
[20]曹丽华,商秀丽,王述森,季春燕,何志义,罗阳.肯尼迪病基因突变分析[J].中国医科大学学报,2011,40(7):577-579Cao L H,Shang X L,Wang S S,Ji C Y,He Z Y,Luo Y.Analysis of gene mutation on patient with Kennedy's disease[J].Journal of China Medical University,2011,40(7):577-579(in Chinese)
[21]Xin J G,Yang H Q,Fan N N,Zhao B T,Ouyang Z,Liu Z M,Zhao Y,Li X P,Song J,Yang Y,Zou Q J,Yan Q M,Zeng Y Z,Lai L X.Highly efficient generation of GGTA1 biallelic knockout inbred mini-pigs with TALENs[J].Plos One.2013,8(12):e84250
[22]Lu B,Smock S L,Castleberry T A,Owen T A.Molecular cloning and functional characterization of the canine androgen receptor[J].Molecular and Cellular Biochemistry,2001,226(1):129-140
[23]秦立红,刘伟英,张丽颖,张金玉,张国梁,赵玉民,张嘉保,赵志辉.草原红牛不同组织雄激素受体基因表达量检测及比较研究[J].中国兽医学报,2010,30(4):544-548Qin L H,Liu W R,Zhang L Y,Zhang J Y,Zhang G L,Zhao Y M,Zhang J B,Zhao Z H.Expression and comparative study on androgen receptor(AR)gene in different tissues of Chinese red steppes[J].China Journal of Veterinary Science,2010,30(4):544-548(in Chinese)
[2]Baker M E.Steroid receptor phylogeny and vertebrate origins[J].Molecular&Cellular Endocrinology,1997,135(2):101-107
[3]Zhang C X,Yeh S Y,Chen Y T,Wu C C,Chuang K H,Lin H Y,Wang R S,Chang Y J,Mendis-Handagama C,Hu L Q,Lardy H,Chang C.Oligozoospermia with normal fertility in male mice lacking the androgen receptor in testis peritubular myoid cells[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(47):17718-17723
[4]Xu Q Q,Lin H Y,Yeh S D,Yu I C,Wang R S,Chen Y T,Zhang C X,Altuwaijri S,Chen L M,Chuang K H,Chiang H S,Yeh S Y,Chang C S.Infertility with defective spermatogenesis and steroidogenesis in male mice lacking androgen receptor in Leydig cells[J].Endocrine,2007,32(1):96-106
[5]Kumar R,Johnson B H,Thompson E B.Overview of the structural basis for transcription regulation by nuclear hormone receptors[J].Essays in Biochemistry,2004,40(1):27-39
[6]黄宝锋,刘智皓,吴风瑞,王德寿.雄激素受体的结构和功能[J].四川动物,2008,27(2):307-310Huang B F,Liu Z H,Wu F R,Wang D S.The structure and function of androgen receptor[J].Sichuan Journal of Zoology,2008,27(2):307-310(in Chinese)
[7]Wang Q,Li W,Liu X S,Carroll J S,Janne O A,Keeton E K,Chinnaiyan A M,Pienta K J,Brown M.A hierarchical network of transcription factors governs androgen receptor-dependent prostate cancer growth[J].Molecular Cell,2007,27(27):380-392
[8]Chang C S,Kokontis J,Liao S T.Molecular cloning of human and rat complementary DNA encoding androgen receptors[J].Science,1988,240(240):324-326
[9]Trapman J,Klaassen P,Kuiper G G,van der Korput J A,Faber P W,van Rooij H C,Geurts van Kessel A,Voorhorst M M,Mulder E,Brinkmann A O.Cloning,structure and expression of a cDNA encoding the human androgen receptor[J].Biochemical&Biophysical Research Communications,1988,153(1):241-248
[10]夏欣一,杨滨,崔英霞,黄宇烽.男性不育的遗传学病因研究进展[J].中华男科学杂志,2008,14(9):837-841Xia X Y,Yang B,Cui Y X,Huang Y F.Genetic cause of male infertility[J].National Journal of Andrology,2008,14(9):837-841(in Chinese)
[11]Yu P,Zhang L,Li S,Li Y,Cheng J,Lu Y,Zeng Y,Bu H.Screening and analysis of porcine endogenous retrovirus in Chinese Banna minipig inbred line[J].Transplant Proceedings,2004,36(8):2485-2487
[12]Zeng R,Zeng Y Z.Molecular cloning and characterization of SLA-DR genes in the 133-family of the Banna mini-pig inbred line[J].Animal Genetics,2005,36(3):267-269
[13]Huo J L,Wang P,Zhao Y,Huo H L,Liu L X,Wang R,Xiao H,Zeng Y Z.Molecular cloning,mRNA expression and characterization of a novel FAIM1gene from Chinese Banna mini-pig inbred line(BMI)[J].Journal of Animal and Veterinary Advances,2012,11(8):1080-1086
[14]Wang P,Wang S Y,Qin C Y,Huo J L.Characterization and expression of PGK2and PHKG2in testis tissues from fertile and sterile Banna mini-pig inbred line(BMI)[J].Research Journal of Biotechnology,2015,10(2):28-35
[15]陈园园,潘伟荣,霍金龙,王淑燕,王配,查星琴,曾养志.版纳微型猪近交系不育和可育公猪CRISP2基因全长编码区序列克隆及睾丸差异表达分析[J].云南农业大学学报自然科学,2015,30(5):698-705Chen Y Y,Pan W R,Huo J L,Wang S Y,Wang P,Zha X Q,Zeng Y Z.Cloning and testis expression difference analysis of coding region sequence of CRISP2gene in sterile and fertile boar of Banna mini-pig inbred line[J].Journal of Yunnan Agricultural University:Natural Science,2015,30(5):698-705(in Chinese)
[16]Florea L,Hartzell G,Zhang Z,Rubin G M,Miller W.A computer program for aligning a cDNA sequence with a genomic DNA sequence[J].Genome Research,1998,8(9):967-974
[17]郭安源,朱其慧,陈新,罗静初.GSDS:基因结构显示系统[J].遗传,2007,29(8):1023-1026Guo A Y,Zhu Q H,Chen X,Luo J C.GSDS:A gene structure display server[J].Hereditas,2007,29(8):1023-1026(in Chinese)
[18]Pu L,Han K,Xie F,Zou Z,Close D,Zhang Z,Wang Y.Molecular cloning,characterization,and gene expression of the androgen receptor in the large yellow croaker,Larimichthys crocea[J].Fish Physiology and Biochemistry,2013,39(2):309-324
[19]Simental J A,Sar M,Lane M V,French F S,Wilson E M.Transcriptional activation and nuclear targeting signals of the human androgen receptor[J].Journal of Biological Chemistry,1991,266(1):510-518
[20]曹丽华,商秀丽,王述森,季春燕,何志义,罗阳.肯尼迪病基因突变分析[J].中国医科大学学报,2011,40(7):577-579Cao L H,Shang X L,Wang S S,Ji C Y,He Z Y,Luo Y.Analysis of gene mutation on patient with Kennedy's disease[J].Journal of China Medical University,2011,40(7):577-579(in Chinese)
[21]Xin J G,Yang H Q,Fan N N,Zhao B T,Ouyang Z,Liu Z M,Zhao Y,Li X P,Song J,Yang Y,Zou Q J,Yan Q M,Zeng Y Z,Lai L X.Highly efficient generation of GGTA1 biallelic knockout inbred mini-pigs with TALENs[J].Plos One.2013,8(12):e84250
[22]Lu B,Smock S L,Castleberry T A,Owen T A.Molecular cloning and functional characterization of the canine androgen receptor[J].Molecular and Cellular Biochemistry,2001,226(1):129-140
[23]秦立红,刘伟英,张丽颖,张金玉,张国梁,赵玉民,张嘉保,赵志辉.草原红牛不同组织雄激素受体基因表达量检测及比较研究[J].中国兽医学报,2010,30(4):544-548Qin L H,Liu W R,Zhang L Y,Zhang J Y,Zhang G L,Zhao Y M,Zhang J B,Zhao Z H.Expression and comparative study on androgen receptor(AR)gene in different tissues of Chinese red steppes[J].China Journal of Veterinary Science,2010,30(4):544-548(in Chinese)