撒坝猪SPDEF基因克隆及其生物信息学分析
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摘要
为明确猪SPDEF基因的遗传特性,试验对不同猪种SPDEF基因的CDS区进行序列扩增、测序、拼接,并对撒坝猪SPDEF基因CDS序列进行生物信息学分析。采用PCR直接测序法测定猪SPDEF基因外显子序列,运用SeqMan进行序列拼接。采用生物信息学软件分析撒坝猪SPDEF基因开放阅读框及其编码蛋白的基本理化性质、疏水性、信号肽、跨膜区、亚细胞定位、磷酸化位点、糖基化位点、二级结构、三级结构、功能结构域,并构建系统进化树。结果表明,各猪种SPDEF基因编码区仅在外显子1、2分别检测到1个同义突变(C13971T、C16541T),表明该基因编码区在所检测的猪群中高度保守。生物信息学分析发现,该基因有1个长1 092 bp的完整开放阅读框;SPDEF蛋白中丝氨酸(Ser)含量最高(10.7%),体外理论半衰期为30 h,属于不稳定的亲水性蛋白;该蛋白不存在信号肽及跨膜结构,存在39个潜在的磷酸化位点和1个糖基化位点,亚细胞定位于细胞质;二级结构预测中,参与无规则卷曲的氨基酸最多(55.10%),其次是α-螺旋(28.37%);功能结构域预测发现,该蛋白存在1个ETS功能结构域和1个SAM-PNT功能结构域。系统进化树结果显示,撒坝猪与牛、绵羊和山羊的亲缘关系较近。本研究为进一步分析撒坝猪SPDEF基因的功能奠定了一定的理论基础。
In order to identify the genetic characteristics of the SPDEF gene,the CDS of different pig breeds were amplified,sequenced,spliced,and the CDS of Saba pigs were analyzed by bioinformatics.The exon sequence of pigs were detected by direct sequencing after PCR,and sequence splicing were performed using SeqMan.The open reading frame(ORF) of SPDEF gene,and basic physicochemical properties,hydrophobicity,signal peptide,transmembrane region,subcellular localization,phosphorylation site,glycosylation site,secondary structure,tertiary structure,functional domain of SPDEF protein were predicted,and phylogenetic tree was constructed using bioinformatics software.The results showed that only 2 synonymous SNPs were detected in exon 1(C13971 T) and exon 2(C16541 T) of SPDEF gene in different pigs,respectively,indicating that the coding region of SPDEF gene was highly conserved among the tested herds.SPDEF gene had a complete ORF at length of 1 092 bp.The Ser content was the highest,which accounted for 10.7%.SPDEF was an unstable hydrophilic protein with an estimated half-life of 30 h in vitro.The SPDEF protein has no signal peptide and transmembrane structure,and had 39 potential phosphorylation sites and 1 glycosylation site.The protein located the cytoplasm in analysis of subcellular localization.In the secondary structure prediction,the amino acids involved in the random coil were the most,accounting for 55.10%,followed by the α-helix region,accounting for 28.37%.Functional domain predictions revealed the existence of an ETS functional domain and an SAM-PNT functional domain.Sequence homology analysis showed that pig has higher homology with Bos taurus,Ovis aries and Capra hircus.This study could provide references for further analysis of the function of SPDEF gene in Saba pigs.
In order to identify the genetic characteristics of the SPDEF gene,the CDS of different pig breeds were amplified,sequenced,spliced,and the CDS of Saba pigs were analyzed by bioinformatics.The exon sequence of pigs were detected by direct sequencing after PCR,and sequence splicing were performed using SeqMan.The open reading frame(ORF) of SPDEF gene,and basic physicochemical properties,hydrophobicity,signal peptide,transmembrane region,subcellular localization,phosphorylation site,glycosylation site,secondary structure,tertiary structure,functional domain of SPDEF protein were predicted,and phylogenetic tree was constructed using bioinformatics software.The results showed that only 2 synonymous SNPs were detected in exon 1(C13971 T) and exon 2(C16541 T) of SPDEF gene in different pigs,respectively,indicating that the coding region of SPDEF gene was highly conserved among the tested herds.SPDEF gene had a complete ORF at length of 1 092 bp.The Ser content was the highest,which accounted for 10.7%.SPDEF was an unstable hydrophilic protein with an estimated half-life of 30 h in vitro.The SPDEF protein has no signal peptide and transmembrane structure,and had 39 potential phosphorylation sites and 1 glycosylation site.The protein located the cytoplasm in analysis of subcellular localization.In the secondary structure prediction,the amino acids involved in the random coil were the most,accounting for 55.10%,followed by the α-helix region,accounting for 28.37%.Functional domain predictions revealed the existence of an ETS functional domain and an SAM-PNT functional domain.Sequence homology analysis showed that pig has higher homology with Bos taurus,Ovis aries and Capra hircus.This study could provide references for further analysis of the function of SPDEF gene in Saba pigs.
引文
[1] WU J,QIN W,WANG Y,et al.SPDEF is overexpressed in gastric cancer and triggers cell proliferation by forming a positive regulation loop with FoxM1[J].Journal of Cellular Biochemistry,2018,1:1-13.
[2] HSU T,TROJANOWSKA M,DENNIS K W.ETS proteins in biological control and cancer[J].Journal of Cellular Biochemistry,2004,91(5):896-903.
[3] KAR A,HARTMANN A G.Molecular mechanisms of ETS transcription factor-mediated tumorigenesis[J].Critical Reviews in Biochemistry and Molecular Biology,2013,48(6):522-543.
[4] SHARROCKS A D,BROWN A L,YAN L,et al.The ETS-domain transcription factor family[J].International Journal of Biochemistry & Cell Biology,1997,29(12):1371-1387.
[5] OETTGEN P,EDUARDO F,SUN Z J,et al.PDEF,a novel prostate epithelium-specific ets transcription factor,interacts with the androgen receptor and activates prostate-specific antigen gene expression [J].The Journal of Biological Chemistry,2000,275(2):1216-1225.
[6] STEFFAN J J,KOUL S,RANDALL B M,et al.The transcription factor SPDEF suppresses prostate tumor metastasis[J].The Journal of Biological Chemistry,2012,287(35):22997-29968.
[7] HARTMANN A G,DUVAL D L,BRADFORD A P.ETS transcription factors in endocrine systems[J].Trends in Endocrinology & Metabolism,2007,18(4):150-158.
[8] CHENG X H,BLACK M,VLADIMIR U,et al.SPDEF inhibits prostate carcinogenesis by disrupting a positive feedback loop in regulation of the Foxm1 oncogene[J].PLoS Genetics,2014,10(9):1-14.
[9] CHEN W Y,TSAI Y C,YEH H L,et al.Loss of SPDEF and gain of TGFBI activity after androgen deprivation therapy promote EMT and bone metastasis of prostate cancer[J].Science Signaling,2017,10(492):6826.
[10] SCHAEFER J S,SABHERWAL Y,HEIDI Y S,et al.Transcriptional regulation of p21/CIP1 cell cycle inhibitor by PDEF controls cell proliferation and mammary tumor progression[J].The Journal of Biological Chemistry,2010,285(15):11126-11258.
[11] GHADERSOHI A,PAN D L,FAYAZI Z,et al.Prostate-derived Ets transcription factor (PDEF) downregulates survivin expression and inhibits breast cancer cell growth in vitro and xenograft tumor formation in vivo[J].Breast Cancer RES Treat,2007,102(1):19-30.
[12] MOUSSA O,TURNER D P,FELDMAN R J,et al.PDEF is a negative regulator of colon cancer cell growth and migration[J].Journal of Cellular Biochemistry,2009,108(6):1389-1398.
[13] HUNG L Y,TAEKO K N,CHEN M S,et al.SPDEF induces quiescence of colorectal cancer cells by changing the transcriptional targets of β-catenin[J].Gastroenterology,2017,153(1):205-218.
[14] GREGORIEFF A,DANIEL E S,KUJALA P,et al.The ets-domain transcription factor Spdef promotes maturation of goblet and paneth cells in the intestinal epithelium[J].Gastroenterology,2009,137(4):1333-1345.
[15] ARONSON B E,STAPLETON K A,VISSERS L A,et al.Spdef deletion rescues the crypt cell proliferation defect in conditional Gata6 null mouse small intestine[J].BMC Molecular Biology,2014,2014,15(1):3-12.
[16] WANG L G,ZHANG L C,HUA Y,et al.Genome-wide association studies identify the loci for 5 exterior traits in a Large White×Minzhu pig population[J].PLoS One,2014,9(8):e103766.
[17] SUN H,WANG Z,ZHANG Z,et al.Genomic signatures reveal selection of characteristics within and between Meishan pig populations[J].Animal Genetics,2018,49(2):119-126.
[18] 连林生,鲁绍雄.撒坝猪血清酯酶多态性与繁殖性能关系的研究[J].遗传,1999,21(4):25-28.LIAN L S,LU S X.A study of the relationship between serum esterase polymorphism and reproductive performance of saba pig[J].Hereditas,1999,21(4):25-28.(in Chinese)
[19] 丁艳,鲁绍雄,连林生.撒坝猪胰岛素样生长因子-Ⅰ基因多态性及其与部分生长性状的关联分析[J].中国畜牧兽医,2012,39(10):181-184.DING Y,LU S X,LIAN L S.The polymorphism of IGF-Ⅰ gene and its relationship with some growth traits in Saba pig[J].China Animal Husbandry & Veterinary Medicine,2012,39(10):181-184.(in Chinese)
[20] 罗铮,翁吉梅,封竣琪,等.黔北麻羊MEF2D基因多态性及生物信息学分析[J].中国畜牧兽医,2018,45(10):2661-2669.LUO Z,WENG J M,FENG J Q,et al.Polymorphism and bioinformatics analysis of MEF2D gene in Qianbei Ma goat[J].China Animal Husbandry & Medicine,2018,45(10):2661-2669.(in Chinese)
[21] 王明亮,陈鹏,郝明超,等.黄牛FSHR基因的生物信息学分析[J].甘肃农业大学学报,2012,47(4):7-13.WANG M L,CHEN P,HAO M C,et al.Bioinformatics analysis of cattle FSHR gene[J].Journal of Gansu Agricultural University,2012,47(4):7-13.(in Chinese)
[22] LAWRENCE A K,STERNBERG M E.protein structure prediction on the Web:A case study using the Phyre server[J].Nature Protocols,2009,4(3):363-367.
[23] SEMENTCHENKO V I,DENNIS K W.ETS target genes past present and future[J].Oncogene,2000,19(55):6533-6548.
[24] RODRIGO E T,JULIANO D P,KRISTAL C D,et al.GADD45α and γ interaction with CDK11p58 regulates SPDEF protein stability and SPDEF-mediated effects on cancer cell migration [J].Oncotarget,2016,7(12):11387-13865.
[25] SONG J,HEIJINK I H,KISTEMAKER L M,et al.Aberrant DNA methylation and expression of SPDEF and FOXA2 in airway epithelium of patients with COPD[J].Clinical Epigenetics,2017,9,42.
[2] HSU T,TROJANOWSKA M,DENNIS K W.ETS proteins in biological control and cancer[J].Journal of Cellular Biochemistry,2004,91(5):896-903.
[3] KAR A,HARTMANN A G.Molecular mechanisms of ETS transcription factor-mediated tumorigenesis[J].Critical Reviews in Biochemistry and Molecular Biology,2013,48(6):522-543.
[4] SHARROCKS A D,BROWN A L,YAN L,et al.The ETS-domain transcription factor family[J].International Journal of Biochemistry & Cell Biology,1997,29(12):1371-1387.
[5] OETTGEN P,EDUARDO F,SUN Z J,et al.PDEF,a novel prostate epithelium-specific ets transcription factor,interacts with the androgen receptor and activates prostate-specific antigen gene expression [J].The Journal of Biological Chemistry,2000,275(2):1216-1225.
[6] STEFFAN J J,KOUL S,RANDALL B M,et al.The transcription factor SPDEF suppresses prostate tumor metastasis[J].The Journal of Biological Chemistry,2012,287(35):22997-29968.
[7] HARTMANN A G,DUVAL D L,BRADFORD A P.ETS transcription factors in endocrine systems[J].Trends in Endocrinology & Metabolism,2007,18(4):150-158.
[8] CHENG X H,BLACK M,VLADIMIR U,et al.SPDEF inhibits prostate carcinogenesis by disrupting a positive feedback loop in regulation of the Foxm1 oncogene[J].PLoS Genetics,2014,10(9):1-14.
[9] CHEN W Y,TSAI Y C,YEH H L,et al.Loss of SPDEF and gain of TGFBI activity after androgen deprivation therapy promote EMT and bone metastasis of prostate cancer[J].Science Signaling,2017,10(492):6826.
[10] SCHAEFER J S,SABHERWAL Y,HEIDI Y S,et al.Transcriptional regulation of p21/CIP1 cell cycle inhibitor by PDEF controls cell proliferation and mammary tumor progression[J].The Journal of Biological Chemistry,2010,285(15):11126-11258.
[11] GHADERSOHI A,PAN D L,FAYAZI Z,et al.Prostate-derived Ets transcription factor (PDEF) downregulates survivin expression and inhibits breast cancer cell growth in vitro and xenograft tumor formation in vivo[J].Breast Cancer RES Treat,2007,102(1):19-30.
[12] MOUSSA O,TURNER D P,FELDMAN R J,et al.PDEF is a negative regulator of colon cancer cell growth and migration[J].Journal of Cellular Biochemistry,2009,108(6):1389-1398.
[13] HUNG L Y,TAEKO K N,CHEN M S,et al.SPDEF induces quiescence of colorectal cancer cells by changing the transcriptional targets of β-catenin[J].Gastroenterology,2017,153(1):205-218.
[14] GREGORIEFF A,DANIEL E S,KUJALA P,et al.The ets-domain transcription factor Spdef promotes maturation of goblet and paneth cells in the intestinal epithelium[J].Gastroenterology,2009,137(4):1333-1345.
[15] ARONSON B E,STAPLETON K A,VISSERS L A,et al.Spdef deletion rescues the crypt cell proliferation defect in conditional Gata6 null mouse small intestine[J].BMC Molecular Biology,2014,2014,15(1):3-12.
[16] WANG L G,ZHANG L C,HUA Y,et al.Genome-wide association studies identify the loci for 5 exterior traits in a Large White×Minzhu pig population[J].PLoS One,2014,9(8):e103766.
[17] SUN H,WANG Z,ZHANG Z,et al.Genomic signatures reveal selection of characteristics within and between Meishan pig populations[J].Animal Genetics,2018,49(2):119-126.
[18] 连林生,鲁绍雄.撒坝猪血清酯酶多态性与繁殖性能关系的研究[J].遗传,1999,21(4):25-28.LIAN L S,LU S X.A study of the relationship between serum esterase polymorphism and reproductive performance of saba pig[J].Hereditas,1999,21(4):25-28.(in Chinese)
[19] 丁艳,鲁绍雄,连林生.撒坝猪胰岛素样生长因子-Ⅰ基因多态性及其与部分生长性状的关联分析[J].中国畜牧兽医,2012,39(10):181-184.DING Y,LU S X,LIAN L S.The polymorphism of IGF-Ⅰ gene and its relationship with some growth traits in Saba pig[J].China Animal Husbandry & Veterinary Medicine,2012,39(10):181-184.(in Chinese)
[20] 罗铮,翁吉梅,封竣琪,等.黔北麻羊MEF2D基因多态性及生物信息学分析[J].中国畜牧兽医,2018,45(10):2661-2669.LUO Z,WENG J M,FENG J Q,et al.Polymorphism and bioinformatics analysis of MEF2D gene in Qianbei Ma goat[J].China Animal Husbandry & Medicine,2018,45(10):2661-2669.(in Chinese)
[21] 王明亮,陈鹏,郝明超,等.黄牛FSHR基因的生物信息学分析[J].甘肃农业大学学报,2012,47(4):7-13.WANG M L,CHEN P,HAO M C,et al.Bioinformatics analysis of cattle FSHR gene[J].Journal of Gansu Agricultural University,2012,47(4):7-13.(in Chinese)
[22] LAWRENCE A K,STERNBERG M E.protein structure prediction on the Web:A case study using the Phyre server[J].Nature Protocols,2009,4(3):363-367.
[23] SEMENTCHENKO V I,DENNIS K W.ETS target genes past present and future[J].Oncogene,2000,19(55):6533-6548.
[24] RODRIGO E T,JULIANO D P,KRISTAL C D,et al.GADD45α and γ interaction with CDK11p58 regulates SPDEF protein stability and SPDEF-mediated effects on cancer cell migration [J].Oncotarget,2016,7(12):11387-13865.
[25] SONG J,HEIJINK I H,KISTEMAKER L M,et al.Aberrant DNA methylation and expression of SPDEF and FOXA2 in airway epithelium of patients with COPD[J].Clinical Epigenetics,2017,9,42.
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