猪LPAR3基因克隆及其在子宫内膜细胞的表达
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摘要
【目的】获得猪LPAR3基因完整mRNA及基因结构,研究其启动子活性;探究LPAR3基因在子宫内膜的转录调控及可能影响母猪产仔的机制。【方法】应用5'RACE和3'RACE技术获取LPAR3基因完整mRNA序列;预测5'调控区潜在的启动子转录因子结合位点及CpG岛,构建不同长度的启动子双荧光素酶报告基因重组载体,与pRL-TK质粒共同转染至猪子宫内膜细胞,检测启动子活性;应用RT-qPCR比较LPAR3基因在妊娠第12天的二花脸猪和长大二元猪子宫内膜的相对表达量;应用亚硫酸氢钠修饰后测序比较LPAR3基因在妊娠第12天的二花脸猪和长大二元猪子宫内膜的甲基化状态。【结果】猪LPAR3 mRNA全长为2 127 bp,其中5'UTR和3'UTR的长度分别为202和860 bp,CDS区为1 065 bp。克隆获得包括LPAR3转录起始位点上游3 080 bp (–2 430/+650bp)的5'调控序列,分析预测显示该调控区不存在TATA box,存在GC元件、CPBP及糖皮质激素受体IR3等调控因子结合位点,且在–190/–84和–44/+651 bp处存在2个潜在CpG岛。成功构建9个不同长度的5'缺失报告重组载体并转染猪子宫内膜细胞。双荧光素酶活性检测结果显示,启动子P4(+454/+80 bp)的转录活性最高,其次是P6(–123/+80 bp)。RT-qPCR结果显示,妊娠第12天二花脸猪LPAR3基因在子宫内膜的表达量高于在其他组织的表达量,且极显著高于在妊娠第12天长大二元猪子宫内膜的表达量,LPAR3在2个猪种子宫内膜均处于低甲基化状态且差异不显著。【结论】猪LPAR3 mRNA全长为2 127 bp,妊娠第12天LAPR3基因在二花脸猪子宫内膜表达高于其在长大二元猪子宫内膜的表达,显示LPAR3可能参与了猪早期妊娠并影响产仔数。
【Objective】To obtain the complete mRNA sequence and structure of porcine LPAR3 gene, study the gene promoter activity, and explore the transcriptional regulation of LPAR3 gene in endometrium and the mechanisms which may affect sow farrowing. 【Method】The 5'RACE and 3'RACE techniques were used to obtain the complete LPAR3 mRNA sequence. The potential promoter transcription factor binding sites and CpG islands in the 5' regulatory region were predicted. Recombinant vectors with different length of promoters and dual-luciferase reporter gene were constructed, then were transfected with pRL-TK plasmid into pig endometrial cells, and the promoter activities were detected. RT-qPCR was used to compare the relative expression of LPAR3 gene in Erhualian(ER) and Landrace×Large White(LL) pigs on the 12 th day of gestation. Sodium bisulfite modified sequencing was used to compare the methylation status of LPAR3 gene in endometrial tissues of ER and LL pigs on the 12 th day of gestation. 【Result】 The full length of pig LPAR3 mRNA was 2 127 bp,the 5'UTR and 3'UTR were 202 and 860 bp respectively, and the CDS region was 1 065 bp. The 5' regulatory sequence, including 3 080 bp(–2 430/+650 bp) upstream of the LPAR3 gene, was cloned. The online prediction results of the 5' regulatory region showed that there was no TATA box in the LPAR3 promoter, but there were GC element and binding sites for CPBP and other regulatory factors. Two potential CpG islands were found at–190/–84 and –44/+651 bp. Nine recombinant vectors with 5' deletions of the promoter were constructed and transfected into pig endometrium cells. The dual-luciferase assay showed that the activity of promoter P4(+454/+80 bp) was the highest, followed by P6(–123/+80 bp). RT-qPCR results showed that the expression of LPAR3 gene in the endometrium of ER pigs on the 12 th day of gestation was higher than that in other tissues,and it was extremely significantly higher than that in the endometrium of LL pigs on the 12 th day of gestation.The methylation status of LPAR3 gene was low in the endometrium of two breeds and had no significant difference between two breeds. 【Conclusion】The full length of pig LPAR3 mRNA is 2 127 bp. The endometrial expression level of LAPR3 gene in ER pigs is higher than that in LL pigs on the 12 th day of gestation, indicating that LPAR3 gene may be involved in early gestation of pigs and affects litter size.
【Objective】To obtain the complete mRNA sequence and structure of porcine LPAR3 gene, study the gene promoter activity, and explore the transcriptional regulation of LPAR3 gene in endometrium and the mechanisms which may affect sow farrowing. 【Method】The 5'RACE and 3'RACE techniques were used to obtain the complete LPAR3 mRNA sequence. The potential promoter transcription factor binding sites and CpG islands in the 5' regulatory region were predicted. Recombinant vectors with different length of promoters and dual-luciferase reporter gene were constructed, then were transfected with pRL-TK plasmid into pig endometrial cells, and the promoter activities were detected. RT-qPCR was used to compare the relative expression of LPAR3 gene in Erhualian(ER) and Landrace×Large White(LL) pigs on the 12 th day of gestation. Sodium bisulfite modified sequencing was used to compare the methylation status of LPAR3 gene in endometrial tissues of ER and LL pigs on the 12 th day of gestation. 【Result】 The full length of pig LPAR3 mRNA was 2 127 bp,the 5'UTR and 3'UTR were 202 and 860 bp respectively, and the CDS region was 1 065 bp. The 5' regulatory sequence, including 3 080 bp(–2 430/+650 bp) upstream of the LPAR3 gene, was cloned. The online prediction results of the 5' regulatory region showed that there was no TATA box in the LPAR3 promoter, but there were GC element and binding sites for CPBP and other regulatory factors. Two potential CpG islands were found at–190/–84 and –44/+651 bp. Nine recombinant vectors with 5' deletions of the promoter were constructed and transfected into pig endometrium cells. The dual-luciferase assay showed that the activity of promoter P4(+454/+80 bp) was the highest, followed by P6(–123/+80 bp). RT-qPCR results showed that the expression of LPAR3 gene in the endometrium of ER pigs on the 12 th day of gestation was higher than that in other tissues,and it was extremely significantly higher than that in the endometrium of LL pigs on the 12 th day of gestation.The methylation status of LPAR3 gene was low in the endometrium of two breeds and had no significant difference between two breeds. 【Conclusion】The full length of pig LPAR3 mRNA is 2 127 bp. The endometrial expression level of LAPR3 gene in ER pigs is higher than that in LL pigs on the 12 th day of gestation, indicating that LPAR3 gene may be involved in early gestation of pigs and affects litter size.
引文
[1]TAYADE C,FANG Y,CROY B A.A review of gene expression in porcine endometrial lymphocytes,endothelium and trophoblast during pregnancy success and failure[J].J Reprod Dev,2007,53(3):455-463.
[2]洪林君.影响猪胚胎附植及胎盘褶皱发育相关基因的鉴定[D].武汉:华中农业大学,2016.
[3]BENNETT G L,LEYMASTER K A.Integration of ovulation rate,potential embryonic viability and uterine capacity into a model of litter size in swine[J].J Anim Sci,1989,67(5):1230-1241.
[4]FOXCROFT G R,DIXON W T,NOVAK S,et al.The biological basis for prenatal programming of postnatal performance in pigs[J].J Anim Sci,2006,84(Suppl):E105-E112.
[5]葛云山,徐金友,刘明智,等.二花脸猪繁殖性能的观察[J].江苏农业科学,1982(4):40-43.
[6]杜红丽,陈静,张玉山,等.二花脸与杜洛克猪繁殖相关基因表达差异[J].华南农业大学学报,2008,29(2):99-103.
[7]KIM M,SEO H,CHOI Y,et al.Microarray analysis of gene expression in the uterine endometrium during the implantation period in pigs[J].Asian-australas J Anim Sci,2012,25(8):1102-1116.
[8]王首奇.母猪怀孕11-12天子宫内膜差异表达基因的筛选与分析[D].广州:华南农业大学,2011.
[9]SEO H,CHOI Y,SHIM J,et al.Analysis of the lysophosphatidic acid-generating enzyme ENPP2 in the uterus during pregnancy in pigs[J].Biol Reprod,2012,87(4):77.
[10]MAUCO G,CHAP H,SIMON M F,et al.Phosphatidic and lysophosphatidic acid production in phospholipase C-and thrombin-treated platelets:Possible involvement of a platelet lipase[J].Biochimie,1978,60(6/7):653-661.
[11]YE X,CHUN J.Lysophosphatidic acid(LPA)signaling in vertebrate reproduction[J].Trends Endocrinol Metab,2010,21(1):17-24.
[12]王溦.LPAR3、COX-2和VEGF蛋白在反复移植失败患者着床期子宫内膜表达的研究[D].郑州:郑州大学,2014.
[13]CHA J,SUN X,DEY S K.Mechanisms of implantation:Strategies for successful pregnancy[J].Nat Med,2012,18(12):1754-1767.
[14]郭燕红,张雷,邵素霞,等.溶血磷脂酸受体3在小鼠胚胎围着床期子宫内膜的表达及意义[J].解剖学报,2009,40(1):141-145.
[15]SHAH B H,CATT K J.Roles of LPA3 and COX-2 in implantation[J].Trends Endocrinol Metab,2005,16(9):397-399.
[16]DIAO H,LI R,EI ZOWALATY A E,et al.Deletion of lysophosphatidic acid receptor 3(Lpar3)disrupts fine local balance of progesterone and estrogen signaling in mouse uterus during implantation[J].Biol Reprod,2015,93(5):123.
[17]HAMA K,AOKI J,INOUE A,et al.Embryo spacing and implantation timing are differentially regulated by LPA3-mediated lysophosphatidic acid signaling in mice[J].Biol Reprod,2007,77(6):954-959.
[18]AIKAWA S,KANO K,INOUE A,et al.Autotaxin-lysophosphatidic acid-LPA3 signaling at the embryo-epithelial boundary controls decidualization pathways[J].Embo J,2017,36(14):2146-2160.
[19]YE X,HAMA K,CONTOS J J,et al.LPA3-mediated lysophosphatidic acid signaling in embryo implantation and spacing[J].Nature,2005,435(7038):104-108.
[20]李巍巍,秦伟,耿爱华,等.LPAR3、HOXA-11在IVF患者子宫内膜种植窗期的表达及意义[J].中国妇幼保健,2014,29(12):1922-1926.
[21]SHEN J,ZHOU C,ZHU S,et al.Comparative transcrip-tome analysis reveals early pregnancy-specific genes expressed in peripheral blood of pregnant sows[J].PLoSOne,2014,9(12):e114036.
[22]KAMINSKA K,WASIELAK M,BOGACKA,et al.Quantitative expression of lysophosphatidic acid receptor 3 gene in porcine endometrium during the periimplantation period and estrous cycle[J].Prostag Oth Lipid M,2008,85(1/2):26-32.
[23]DEGRELLE S A,BLOMBERG L A,GARRETT W M,et al.Comparative proteomic and regulatory network analyses of the elongating pig conceptus[J].Proteomics,2009,9(10):2678-2694.
[24]KNIGHT J W.Aspects of placental estrogen synthesis in the pig[J].Exp Clin Endocrinol,1994,102(3):175-184.
[25]SEO H,HIM M,CHOI Y,et al.Analysis of lysophosphatidic acid(LPA)receptor and LPA-induced endometrial prostaglandin-endoperoxide synthase 2 expression in the porcine uterus[J].Endocrinology,2008,149(12):6166-6175.
[26]高萍,钟玉宜,张爱玲.母猪卵巢颗粒细胞的分离培养及鉴定[J].广东农业科学,2014,41(4):131-135.
[27]徐慧颖,李娜,张云山.胚胎植入:子宫内膜容受性是关键[J].生殖医学杂志,2014,23(3):198-202.
[28]BOYES J,BIRD A.Repression of genes by DNAmethylation depends on CpG density and promoter strength:Evidence for involvement of a methyl-CpGbinding protein[J].Embo J,1992,11(1):327-333.
[29]KIM M,SEO H,CHOI Y,et al.,Analysis of stage-specific gene expression profiles in the uterine endometrium during pregnancy in pigs[J].PLoS One,2015,10(11):e0143436.
[30]NAMOUS H,PE?AGARICANO F,DEL CORVO M,et al.Integrative analysis of methylomic and transcriptomic data in fetal sheep muscle tissues in response to maternal diet during pregnancy[J].BMC Genomics,2018,19(1):123.
[31]辛鹏慧,岳永莉,李燕,等.牛4种组织中ZAR1基因表达及DNA甲基化修饰[J].畜牧与兽医,2009,41(10):40-42.
[32]张永宏.三个地方品种鸡肌肉组织基因组DNA甲基化分析[D].长春:吉林大学,2014.
[33]蒋曹德,邓昌彦,熊远著.DNA甲基化差异对猪生长性状的影响[J].畜牧兽医学报,2005,36(2):105-110.
[34]苏涛,刘敏,侯斌,等.大白猪与梅山猪子宫内膜转录组及甲基化分析[C]//中国生理学会生殖科学专业委员会.中国动物学会生殖生物学分会第2次联合学术年会暨“生殖科学专业委员会第2次学术交流会”和“生殖生物学分会第16次学术年会”论文集.北京:中国生理学会,2017.
[2]洪林君.影响猪胚胎附植及胎盘褶皱发育相关基因的鉴定[D].武汉:华中农业大学,2016.
[3]BENNETT G L,LEYMASTER K A.Integration of ovulation rate,potential embryonic viability and uterine capacity into a model of litter size in swine[J].J Anim Sci,1989,67(5):1230-1241.
[4]FOXCROFT G R,DIXON W T,NOVAK S,et al.The biological basis for prenatal programming of postnatal performance in pigs[J].J Anim Sci,2006,84(Suppl):E105-E112.
[5]葛云山,徐金友,刘明智,等.二花脸猪繁殖性能的观察[J].江苏农业科学,1982(4):40-43.
[6]杜红丽,陈静,张玉山,等.二花脸与杜洛克猪繁殖相关基因表达差异[J].华南农业大学学报,2008,29(2):99-103.
[7]KIM M,SEO H,CHOI Y,et al.Microarray analysis of gene expression in the uterine endometrium during the implantation period in pigs[J].Asian-australas J Anim Sci,2012,25(8):1102-1116.
[8]王首奇.母猪怀孕11-12天子宫内膜差异表达基因的筛选与分析[D].广州:华南农业大学,2011.
[9]SEO H,CHOI Y,SHIM J,et al.Analysis of the lysophosphatidic acid-generating enzyme ENPP2 in the uterus during pregnancy in pigs[J].Biol Reprod,2012,87(4):77.
[10]MAUCO G,CHAP H,SIMON M F,et al.Phosphatidic and lysophosphatidic acid production in phospholipase C-and thrombin-treated platelets:Possible involvement of a platelet lipase[J].Biochimie,1978,60(6/7):653-661.
[11]YE X,CHUN J.Lysophosphatidic acid(LPA)signaling in vertebrate reproduction[J].Trends Endocrinol Metab,2010,21(1):17-24.
[12]王溦.LPAR3、COX-2和VEGF蛋白在反复移植失败患者着床期子宫内膜表达的研究[D].郑州:郑州大学,2014.
[13]CHA J,SUN X,DEY S K.Mechanisms of implantation:Strategies for successful pregnancy[J].Nat Med,2012,18(12):1754-1767.
[14]郭燕红,张雷,邵素霞,等.溶血磷脂酸受体3在小鼠胚胎围着床期子宫内膜的表达及意义[J].解剖学报,2009,40(1):141-145.
[15]SHAH B H,CATT K J.Roles of LPA3 and COX-2 in implantation[J].Trends Endocrinol Metab,2005,16(9):397-399.
[16]DIAO H,LI R,EI ZOWALATY A E,et al.Deletion of lysophosphatidic acid receptor 3(Lpar3)disrupts fine local balance of progesterone and estrogen signaling in mouse uterus during implantation[J].Biol Reprod,2015,93(5):123.
[17]HAMA K,AOKI J,INOUE A,et al.Embryo spacing and implantation timing are differentially regulated by LPA3-mediated lysophosphatidic acid signaling in mice[J].Biol Reprod,2007,77(6):954-959.
[18]AIKAWA S,KANO K,INOUE A,et al.Autotaxin-lysophosphatidic acid-LPA3 signaling at the embryo-epithelial boundary controls decidualization pathways[J].Embo J,2017,36(14):2146-2160.
[19]YE X,HAMA K,CONTOS J J,et al.LPA3-mediated lysophosphatidic acid signaling in embryo implantation and spacing[J].Nature,2005,435(7038):104-108.
[20]李巍巍,秦伟,耿爱华,等.LPAR3、HOXA-11在IVF患者子宫内膜种植窗期的表达及意义[J].中国妇幼保健,2014,29(12):1922-1926.
[21]SHEN J,ZHOU C,ZHU S,et al.Comparative transcrip-tome analysis reveals early pregnancy-specific genes expressed in peripheral blood of pregnant sows[J].PLoSOne,2014,9(12):e114036.
[22]KAMINSKA K,WASIELAK M,BOGACKA,et al.Quantitative expression of lysophosphatidic acid receptor 3 gene in porcine endometrium during the periimplantation period and estrous cycle[J].Prostag Oth Lipid M,2008,85(1/2):26-32.
[23]DEGRELLE S A,BLOMBERG L A,GARRETT W M,et al.Comparative proteomic and regulatory network analyses of the elongating pig conceptus[J].Proteomics,2009,9(10):2678-2694.
[24]KNIGHT J W.Aspects of placental estrogen synthesis in the pig[J].Exp Clin Endocrinol,1994,102(3):175-184.
[25]SEO H,HIM M,CHOI Y,et al.Analysis of lysophosphatidic acid(LPA)receptor and LPA-induced endometrial prostaglandin-endoperoxide synthase 2 expression in the porcine uterus[J].Endocrinology,2008,149(12):6166-6175.
[26]高萍,钟玉宜,张爱玲.母猪卵巢颗粒细胞的分离培养及鉴定[J].广东农业科学,2014,41(4):131-135.
[27]徐慧颖,李娜,张云山.胚胎植入:子宫内膜容受性是关键[J].生殖医学杂志,2014,23(3):198-202.
[28]BOYES J,BIRD A.Repression of genes by DNAmethylation depends on CpG density and promoter strength:Evidence for involvement of a methyl-CpGbinding protein[J].Embo J,1992,11(1):327-333.
[29]KIM M,SEO H,CHOI Y,et al.,Analysis of stage-specific gene expression profiles in the uterine endometrium during pregnancy in pigs[J].PLoS One,2015,10(11):e0143436.
[30]NAMOUS H,PE?AGARICANO F,DEL CORVO M,et al.Integrative analysis of methylomic and transcriptomic data in fetal sheep muscle tissues in response to maternal diet during pregnancy[J].BMC Genomics,2018,19(1):123.
[31]辛鹏慧,岳永莉,李燕,等.牛4种组织中ZAR1基因表达及DNA甲基化修饰[J].畜牧与兽医,2009,41(10):40-42.
[32]张永宏.三个地方品种鸡肌肉组织基因组DNA甲基化分析[D].长春:吉林大学,2014.
[33]蒋曹德,邓昌彦,熊远著.DNA甲基化差异对猪生长性状的影响[J].畜牧兽医学报,2005,36(2):105-110.
[34]苏涛,刘敏,侯斌,等.大白猪与梅山猪子宫内膜转录组及甲基化分析[C]//中国生理学会生殖科学专业委员会.中国动物学会生殖生物学分会第2次联合学术年会暨“生殖科学专业委员会第2次学术交流会”和“生殖生物学分会第16次学术年会”论文集.北京:中国生理学会,2017.
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