蛋鸡骨钙素基因启动子变异及其转录活性研究
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摘要
骨钙素(osteocalcin, OCN)是由成骨细胞分泌合成的一种非胶原骨基质蛋白,在骨重建、骨矿化等骨代谢过程中发挥重要作用。本研究旨在研究蛋鸡(Gallus gallus domesticus) OCN基因表达调控机制。选取20只50周龄大午粉蛋鸡,提取血液DNA;以Ensembl基因组数据库中OCN基因序列(ENSGALG00000029494)为模板设计引物,扩增OCN基因启动子;利用Sanger测序方法在OCN基因启动子区寻找潜在的遗传变异。采用JASPAR2018在线软件进行转录因子结合位点预测,并构建启动子不同单倍型荧光素酶报告基因重组载体,转染鸡成纤维细胞系DF-1后检测双荧光素酶活性。结果表明,所扩增蛋鸡OCN基因启动子区具有转录活性,含有维生素D受体(Vitamin D receptor, VDR)、RUNX (Runt related transcription factor)1、RUNX2和RUNX3等骨代谢相关转录因子结合位点,测序结果发现在启动子区存在插入片段CCGCACTCTGCACTTTGCGGCCG和缺失片段CCACA,以及C>G和A>G突变,组成4种单倍型;双荧光素酶检测结果表明,启动子区4种单倍型均具有转录活性,但不同单倍型间转录活性存在极显著差异(P<0.01),其中单倍型Ⅳ活性最高。本研究确定了OCN基因核心启动子区,且不同变异类型启动子的转录活性存在差异,为该基因在蛋鸡骨代谢中的转录调控提供了基础资料。
Osteocalcin(OCN) is a kind of non-collagen bone matrix protein secreted and synthesized by osteoblasts, which plays an important role in bone metabolism such as bone remodeling and bone mineralization. This study aimed to investigate the mechanism of gene expression regulation of OCN in laying hens(Gallus gallus domesticus). Twenty 50-week-old DAWUFEN laying hens were selected and the blood was collected for DNA extraction. The primers for promoter region of OCN gene were designed according to the template sequence downloaded from Ensembl database(ENSGALG00000029494). Potential genetic variations in the promoter region of OCN gene were identified by Sanger sequencing. JASPAR2018 was used for transcription factors binding site prediction. The luciferase reporter gene recombinant vectors withdifferent haplotypes were constructed and the dual-luciferase activity was detected by transfecting chicken fibroblast line DF-1. The results showed that the promoter region of the OCN gene in the amplified layer had the transcriptional activity and contained binding sites for bone metabolism-related transcription factors such as Vitamin D receptor(VDR), Runt related transcription factor(RUNX) 1, RUNX2, and RUNX3. Sequencing results revealed the presence of the insert fragment CCGCACTCTGCACTTTGCGGCCG and the deletion fragment CCACA in promoter region, as well as C>G and A>G variations. These variants constituted 4 haplotypes. The dual luciferase assay showed that all the 4 haplotypes in the promoter region had transcriptional activity, but the activity of different haplotypes had extremely significant difference(P<0.01),and the transcriptional activity of haplotype Ⅳ was the highest. In this study, the core promoter region of the OCN gene was identified, and there were differences in the promoter transcriptional activities of different haplotypes. A theoretical basis for the transcriptional regulation of OCN gene could be helpful for bone metabolism of laying hens.
Osteocalcin(OCN) is a kind of non-collagen bone matrix protein secreted and synthesized by osteoblasts, which plays an important role in bone metabolism such as bone remodeling and bone mineralization. This study aimed to investigate the mechanism of gene expression regulation of OCN in laying hens(Gallus gallus domesticus). Twenty 50-week-old DAWUFEN laying hens were selected and the blood was collected for DNA extraction. The primers for promoter region of OCN gene were designed according to the template sequence downloaded from Ensembl database(ENSGALG00000029494). Potential genetic variations in the promoter region of OCN gene were identified by Sanger sequencing. JASPAR2018 was used for transcription factors binding site prediction. The luciferase reporter gene recombinant vectors withdifferent haplotypes were constructed and the dual-luciferase activity was detected by transfecting chicken fibroblast line DF-1. The results showed that the promoter region of the OCN gene in the amplified layer had the transcriptional activity and contained binding sites for bone metabolism-related transcription factors such as Vitamin D receptor(VDR), Runt related transcription factor(RUNX) 1, RUNX2, and RUNX3. Sequencing results revealed the presence of the insert fragment CCGCACTCTGCACTTTGCGGCCG and the deletion fragment CCACA in promoter region, as well as C>G and A>G variations. These variants constituted 4 haplotypes. The dual luciferase assay showed that all the 4 haplotypes in the promoter region had transcriptional activity, but the activity of different haplotypes had extremely significant difference(P<0.01),and the transcriptional activity of haplotype Ⅳ was the highest. In this study, the core promoter region of the OCN gene was identified, and there were differences in the promoter transcriptional activities of different haplotypes. A theoretical basis for the transcriptional regulation of OCN gene could be helpful for bone metabolism of laying hens.
引文
陈伟健,晋大祥,谢炜星,等.2018.RUNX2基因参与骨代谢相关通路的研究进展[J].中国骨质疏松杂志,24(4)557-560.(Chen W J,Jin D X,Xie W X,et al.2018.Advance in the research of Runx2 gene in bone metabolism-related pathway[J].Chinese Journal of Osteoporosis,24(4):557-560.)
成海荣,闫素梅.2009.骨钙素在动物营养中的研究进展[J]饲料博览,22(3):7-10.(Cheng H R,Yan S M.2009.Research progress of osteocalcin in animal nutrition[J].Feed Review,22(3):7-10.)
董雪,侯佳琪,梁旭月,等.2016.骨钙素的研究进展[J].吉林医药学院学报,37(1):67-69.(Dong X,Hou J Q,Liang X Y,et al.2016.Research progress of osteocalcin[J].Journal of Jilin Medical University,37(1):67-69.)
顾丰颖,文生萍,曹振辉,等.2008.维生素D受体基因与骨代谢关系的研究[J].中国畜牧兽医,35(11):44-48.(Gu F Y,Wen S P,Cao Z H,et al.2008.The VDR gene polymorphisms and bone metabolism[J].China Animal Husbandry&Veterinary Medicine,35(11):44-48.)
郭晓强.2011.骨钙素:一种重要的能量代谢调节激素[J].生命科学,23(1):102-105.(Guo X Q.2011.Osteocalcin:An important hormone that regulates energy metabolism[J].Chinese Bulletin of Life Sciences,23(1):102-105.)
吉彩霞,刘晓骅,徐丽,等.2017.RUNX1促进BMP9诱导的间充质干细胞MEFs的成骨分化[J].中国生物工程杂志,37(3):10-17.(Ji C X,Liu X H,Xu L,et al.2017.Runx1 enhances BMP9-induced osteogenic differentiation in mesenchymal stem cell line MEFs[J].China Biotechnology,37(3):10-17.)
李彬,张柳.2009.RUNX2与骨代谢的调控[J].中国骨质疏松杂志,15(1):63-67.(Li B,Zhang L.2009.RUNX2and bone metabolism regulation[J].Chinese Journal of Osteoporosis,15(1):63-67.)
刘冬梅,刘建民.2015.骨钙素对糖代谢的调控作用[J].中华骨质疏松和骨矿盐疾病杂志,8(2):97-102.(Liu D M,Liu J M.2015.Role of osteocalcin in glucose metabolism[J].Chinese Journal of Osteoporosis and Bone Mineral Research,8(2):97-102.)
刘红,廖二元,伍贤平.2004.骨钙素与代谢性骨病[J].国外医学:内分泌学分册,24(4):239-240.(Liu H,Liao E Y,Wu X P,et al.2004.Osteocalcin and metabolic bone disease[J].Foreign Medical Sciences Section of Endocrine,24(4):239-240.)
马利芹,利凯,吕文亭,等.2009.植物雌激素对笼养蛋鸡疲劳综合症骨骼重塑作用机制的研究进展[J].养禽与禽病防治,28(10):2-4.(Ma L Q,Li K,Lv W T,et al.2009.Advances in research on the mechanism of phytoestrogens on bone remodeling in caged hens fatigue syndrome[J].Poultry Husbandry and Disease Control,28(10):2-4.)
沈建军,胡世莲,沈干,等.2010.RUNX3、DAPK基因甲基化及其蛋白表达与胃癌病情的研究[J].安徽医科大学学报,45(5):613-617.(Shen J J,Hu S L,Shen G,et al.2010.Aberrant methylation and protein expression of the RUNX3 and DAPK gene in gastric carcinoma[J].Acta Universitatis Medicinalis Anhui,45(5):613-617.)
王桂华,赵建宁.2011.骨质量的影响因素及其检测方法[J]医学研究生学报,24(10):1095-1098.(Wang G HZhao J N.2011.Influence factors and detection of bone quality[J].Journal of Medical Postgraduates,24(10)1095-1098.)
王志达,夏宇飞,赵岳,等.2017.骨钙素及其对糖代谢影响研究进展[J].中国慢性病预防与控制,25(3):231-233(Wang Z D,Xia Y F,Zhao Y,et al.2017.Progress in osteocalcin and its effects on glucose metabolism[J].Chinese Journal of Prevention and Control of Chronic Diseases,25(3):231-233.)
周向红.2004.维生素D受体基因多态性与骨代谢疾病研究进展[J].医学分子生物学杂志,1(2):107-109.(Zhou XH.2004.Advances in the research of vitamin d receptor gene and bone metabolism-related diseases[J].Foreign Medical Sciences,1(2):107-109.)
Amjad J,George L B,Jasanya B O,et al.2001.Runt homology domain transcription factors(Runx,Cbfa,and AML)mediate repression of the bone sialoprotein promoter Evidence for promoter context-dependent activity of Cbfa proteins[J].Molecular and Cellular Biology,21(8)2891-2905.
Ducy P,Starbuck M,Priemel M,et al.1999.A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development[J].Genes Development,13(8)1025-1036.
Hauschka P V,Lian J B,Gallop P M.1975.Direct identification of the calcium-binding amino-acid,gamma-carboxyglutamate,in mineralized tissue[J].Proceedings of the National Academy of Sciences of the USA,72(10):3925-3929.
Ji C,Liu X H,Xu L,et al.2017.RUNX1 plays an important role in mediating BMP9-induced osteogenic differentiation of mesenchymal stem cells line C3H10T1/2,murine multi-lineage cells lines C2C12 and MEFs[J].International Journal of Molecular Sciences,18(7):1348-1364.
Kayvan Z,Cassie J L,Leonard G.2017.SIRT1 is a positive regulator of the master osteoblast transcription factor,RUNX2[J].PLoS One,12(5):e0178520.
Lee N K,Karsenty G.2008.Reciprocal regulation of bone and energy metabolism[J].Journal of Musculoskeletal Neuronal Interactions,8(4):351.
Lee N K,Sowa H,Hinoi E,et al.2007.Endocrine regulation of energy metabolism by the skeleton[J].Cell,130(3):456-469.
Omri B,Amnon S,Ayako K,et al.2015.Loss of osteoblast Runx3 produces severe congenital osteopenia[J].Molecular and Cellular Biology,35(7):1097-1109.
Oury F,Sumara G,Sumara O,et al.2011.Endocrine regulation of male fertility by the skeleton[J].Cell,144(5):796-809.
Robins S P.1994.Biochemical markers for assessing skeletal growth[J].European Journal of Clinical Nutrition,48(1):199-209.
Smith L B,Saunders P T.2011.The skeleton:The new controller of male fertility[J].Cell,144(5):642-643.
Toshihisa K.2015.The functions of Runx family transcription factors and Cbfb in skeletal development[J].Oral Science International,12(1):1-4.
成海荣,闫素梅.2009.骨钙素在动物营养中的研究进展[J]饲料博览,22(3):7-10.(Cheng H R,Yan S M.2009.Research progress of osteocalcin in animal nutrition[J].Feed Review,22(3):7-10.)
董雪,侯佳琪,梁旭月,等.2016.骨钙素的研究进展[J].吉林医药学院学报,37(1):67-69.(Dong X,Hou J Q,Liang X Y,et al.2016.Research progress of osteocalcin[J].Journal of Jilin Medical University,37(1):67-69.)
顾丰颖,文生萍,曹振辉,等.2008.维生素D受体基因与骨代谢关系的研究[J].中国畜牧兽医,35(11):44-48.(Gu F Y,Wen S P,Cao Z H,et al.2008.The VDR gene polymorphisms and bone metabolism[J].China Animal Husbandry&Veterinary Medicine,35(11):44-48.)
郭晓强.2011.骨钙素:一种重要的能量代谢调节激素[J].生命科学,23(1):102-105.(Guo X Q.2011.Osteocalcin:An important hormone that regulates energy metabolism[J].Chinese Bulletin of Life Sciences,23(1):102-105.)
吉彩霞,刘晓骅,徐丽,等.2017.RUNX1促进BMP9诱导的间充质干细胞MEFs的成骨分化[J].中国生物工程杂志,37(3):10-17.(Ji C X,Liu X H,Xu L,et al.2017.Runx1 enhances BMP9-induced osteogenic differentiation in mesenchymal stem cell line MEFs[J].China Biotechnology,37(3):10-17.)
李彬,张柳.2009.RUNX2与骨代谢的调控[J].中国骨质疏松杂志,15(1):63-67.(Li B,Zhang L.2009.RUNX2and bone metabolism regulation[J].Chinese Journal of Osteoporosis,15(1):63-67.)
刘冬梅,刘建民.2015.骨钙素对糖代谢的调控作用[J].中华骨质疏松和骨矿盐疾病杂志,8(2):97-102.(Liu D M,Liu J M.2015.Role of osteocalcin in glucose metabolism[J].Chinese Journal of Osteoporosis and Bone Mineral Research,8(2):97-102.)
刘红,廖二元,伍贤平.2004.骨钙素与代谢性骨病[J].国外医学:内分泌学分册,24(4):239-240.(Liu H,Liao E Y,Wu X P,et al.2004.Osteocalcin and metabolic bone disease[J].Foreign Medical Sciences Section of Endocrine,24(4):239-240.)
马利芹,利凯,吕文亭,等.2009.植物雌激素对笼养蛋鸡疲劳综合症骨骼重塑作用机制的研究进展[J].养禽与禽病防治,28(10):2-4.(Ma L Q,Li K,Lv W T,et al.2009.Advances in research on the mechanism of phytoestrogens on bone remodeling in caged hens fatigue syndrome[J].Poultry Husbandry and Disease Control,28(10):2-4.)
沈建军,胡世莲,沈干,等.2010.RUNX3、DAPK基因甲基化及其蛋白表达与胃癌病情的研究[J].安徽医科大学学报,45(5):613-617.(Shen J J,Hu S L,Shen G,et al.2010.Aberrant methylation and protein expression of the RUNX3 and DAPK gene in gastric carcinoma[J].Acta Universitatis Medicinalis Anhui,45(5):613-617.)
王桂华,赵建宁.2011.骨质量的影响因素及其检测方法[J]医学研究生学报,24(10):1095-1098.(Wang G HZhao J N.2011.Influence factors and detection of bone quality[J].Journal of Medical Postgraduates,24(10)1095-1098.)
王志达,夏宇飞,赵岳,等.2017.骨钙素及其对糖代谢影响研究进展[J].中国慢性病预防与控制,25(3):231-233(Wang Z D,Xia Y F,Zhao Y,et al.2017.Progress in osteocalcin and its effects on glucose metabolism[J].Chinese Journal of Prevention and Control of Chronic Diseases,25(3):231-233.)
周向红.2004.维生素D受体基因多态性与骨代谢疾病研究进展[J].医学分子生物学杂志,1(2):107-109.(Zhou XH.2004.Advances in the research of vitamin d receptor gene and bone metabolism-related diseases[J].Foreign Medical Sciences,1(2):107-109.)
Amjad J,George L B,Jasanya B O,et al.2001.Runt homology domain transcription factors(Runx,Cbfa,and AML)mediate repression of the bone sialoprotein promoter Evidence for promoter context-dependent activity of Cbfa proteins[J].Molecular and Cellular Biology,21(8)2891-2905.
Ducy P,Starbuck M,Priemel M,et al.1999.A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development[J].Genes Development,13(8)1025-1036.
Hauschka P V,Lian J B,Gallop P M.1975.Direct identification of the calcium-binding amino-acid,gamma-carboxyglutamate,in mineralized tissue[J].Proceedings of the National Academy of Sciences of the USA,72(10):3925-3929.
Ji C,Liu X H,Xu L,et al.2017.RUNX1 plays an important role in mediating BMP9-induced osteogenic differentiation of mesenchymal stem cells line C3H10T1/2,murine multi-lineage cells lines C2C12 and MEFs[J].International Journal of Molecular Sciences,18(7):1348-1364.
Kayvan Z,Cassie J L,Leonard G.2017.SIRT1 is a positive regulator of the master osteoblast transcription factor,RUNX2[J].PLoS One,12(5):e0178520.
Lee N K,Karsenty G.2008.Reciprocal regulation of bone and energy metabolism[J].Journal of Musculoskeletal Neuronal Interactions,8(4):351.
Lee N K,Sowa H,Hinoi E,et al.2007.Endocrine regulation of energy metabolism by the skeleton[J].Cell,130(3):456-469.
Omri B,Amnon S,Ayako K,et al.2015.Loss of osteoblast Runx3 produces severe congenital osteopenia[J].Molecular and Cellular Biology,35(7):1097-1109.
Oury F,Sumara G,Sumara O,et al.2011.Endocrine regulation of male fertility by the skeleton[J].Cell,144(5):796-809.
Robins S P.1994.Biochemical markers for assessing skeletal growth[J].European Journal of Clinical Nutrition,48(1):199-209.
Smith L B,Saunders P T.2011.The skeleton:The new controller of male fertility[J].Cell,144(5):642-643.
Toshihisa K.2015.The functions of Runx family transcription factors and Cbfb in skeletal development[J].Oral Science International,12(1):1-4.