藏绵羊HMOX1基因表达及其多态性与低氧适应性的关联分析
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摘要
血红素加氧酶1(heme oxygenase 1, HMOX1)是血红素加氧酶家族的应激诱导型酶,在机体低氧胁迫代谢调控过程中扮演着重要角色。藏绵羊(Ovis aries)在长期进化过程中形成了独特的低氧适应策略来维持自身的氧平衡。为了探讨HMOX1基因的组织表达及其第三外显子(Exon3)区域多态性与藏绵羊高原低氧适应的关系,以寻找与藏绵羊低氧适应相关的分子标记,本研究以不同海拔地区的藏绵羊和湖羊(对照组)为研究对象,利用q RT-PCR及PCR-单链构象多态性(PCR-single-strand conformation polymorphism, PCR-SSCP)方法检测HMOX1基因的组织表达,以及Exon3区域多态位点,分析不同基因型及等位基因与绵羊血液指标的相关性,以及SNPs与绵羊血液指标的关联性。结果表明,HMOX1基因在藏绵羊和湖羊各组织中均有表达。其中同一品种内,HMOX1在藏绵羊脾脏中高表达(P<0.01),其次在肝、肾脏和背最长肌中表达量也较高(P<0.05);而在湖羊中,HMOX1在肺脏和脂肪中高表达(P<0.01),其次在脾脏、肾脏及肝脏中的表达量也较高(P<0.05)。2个品种间同一组织表达比较结果显示,HMOX1基因在肺、脂肪中的表达量差异极显著(P<0.01)。HMOX1基因Exon3区检测到4个SNPs位点和A、B及C 3个等位基因;通过2个品种间不同基因型比较发现,湖羊的AB型个体血红蛋白含量(hemoglobin, HGB)和红细胞压积(hematokrit, HCT)值明显低于藏绵羊的其他基因型(P<0.01),藏绵羊的AA和BB型个体的HGB和HCT值显著高于AB型(P<0.01),AC型血氧饱和度(oxygen saturation, SO2)值显著高于AA和BB型个体;而c.3775G>A位点上MM基因型个体的SO2、HGB、HCT值显著高于NN基因型(P<0.05)。由此说明AA、BB和AC基因型的c.3775G>A位点变异与血液指标存在相关性,且为G时更有利于对低氧环境的适应。因此,HMOX1基因可作为低氧适应的候选基因,为探索藏绵羊低氧适应分子机制提供基础资料。
Heme oxygenase 1(HMOX1) is a stress-inducible enzyme of the heme oxygenase family.HMOX1 plays an important role in the metabolic regulation of hypoxia stress in the body. Tibetan sheep(Ovisaries)have developed a unique hypoxic adaptation strategy during long-term evolution to maintain their own oxygen balance. In order to investigate the relationship between HMOX1 expression and Exon3 polymorphism and hypoxia adaptation in Tibetan sheep, look for molecular markers relating to hypoxia adaptation, this study used Tibetan sheep and Hu sheep(control group) at different altitudes, the expression of tissues and the genotype polymorphisms in exon3 of HMOX1 gene were detected by qRT-PCR and PCR-single-strand conformation polymorphism(PCR-SSCP), and the associations of different genotypes and alleles with blood parameters of sheep were analyzed. Sexuality and the correlation between different genotypes of single polymorphic loci and blood parameters of sheep were analyzed. The results showed that HMOX1 was expressed in tissues of Tibetan sheep and Hu sheep. In Tibet sheep, the gene was highly expressed in spleen(P<0.01), followed by liver, kidney and longissimus dorsi muscle(P<0.05). In Hu sheep, the gene was highly expressed in lung and fat(P<0.01), followed by liver, kidney and spleen(P<0.05). The expressions of HMOX1 gene in lung and fat tissue were significantly different between Tibetan sheep and Hu sheep(P<0.01).Four SNPs loci and 3 alleles of A, B and C were detected in the Exon3 region of HMOX1 gene. By comparing different genotypes between the two varieties, the hemoglobin(HGB) and hematokrit(HCT) values of AB Hu sheep were significantly lower than those of Tibetan sheep(P<0.01). The HGB and HCT values of BB individuals were significantly higher than those of AB(P<0.01), and the AC-type SO2 values were significantly higher than those of AA and BB individuals and c.3775 G>A sites. The SO2, HGB and HCT values of MM genotypes in c.3775 G>A locus were significantly higher than those of NN genotypes(P<0.05), which indicated that the C. 3775 G>A locus G of AA, BB and AC genotypes was more favorable for adaptation to hypoxic environment. Therefore, HMOX1 gene can be used as candidate gene.These results provide basic data for exploring the molecular mechanism of hypoxia adaptation in Tibetan sheep.
Heme oxygenase 1(HMOX1) is a stress-inducible enzyme of the heme oxygenase family.HMOX1 plays an important role in the metabolic regulation of hypoxia stress in the body. Tibetan sheep(Ovisaries)have developed a unique hypoxic adaptation strategy during long-term evolution to maintain their own oxygen balance. In order to investigate the relationship between HMOX1 expression and Exon3 polymorphism and hypoxia adaptation in Tibetan sheep, look for molecular markers relating to hypoxia adaptation, this study used Tibetan sheep and Hu sheep(control group) at different altitudes, the expression of tissues and the genotype polymorphisms in exon3 of HMOX1 gene were detected by qRT-PCR and PCR-single-strand conformation polymorphism(PCR-SSCP), and the associations of different genotypes and alleles with blood parameters of sheep were analyzed. Sexuality and the correlation between different genotypes of single polymorphic loci and blood parameters of sheep were analyzed. The results showed that HMOX1 was expressed in tissues of Tibetan sheep and Hu sheep. In Tibet sheep, the gene was highly expressed in spleen(P<0.01), followed by liver, kidney and longissimus dorsi muscle(P<0.05). In Hu sheep, the gene was highly expressed in lung and fat(P<0.01), followed by liver, kidney and spleen(P<0.05). The expressions of HMOX1 gene in lung and fat tissue were significantly different between Tibetan sheep and Hu sheep(P<0.01).Four SNPs loci and 3 alleles of A, B and C were detected in the Exon3 region of HMOX1 gene. By comparing different genotypes between the two varieties, the hemoglobin(HGB) and hematokrit(HCT) values of AB Hu sheep were significantly lower than those of Tibetan sheep(P<0.01). The HGB and HCT values of BB individuals were significantly higher than those of AB(P<0.01), and the AC-type SO2 values were significantly higher than those of AA and BB individuals and c.3775 G>A sites. The SO2, HGB and HCT values of MM genotypes in c.3775 G>A locus were significantly higher than those of NN genotypes(P<0.05), which indicated that the C. 3775 G>A locus G of AA, BB and AC genotypes was more favorable for adaptation to hypoxic environment. Therefore, HMOX1 gene can be used as candidate gene.These results provide basic data for exploring the molecular mechanism of hypoxia adaptation in Tibetan sheep.
引文
包鹏甲,裴杰,阎萍,等,2014.藏羊EPO基因遗传多样性与高原低氧适应性[J].江苏农业学报,30(3):581-585.(Bao P J,Pei J,Yan P,et al.2014.EPO gene polymorphism and high altitude hypoxia adaptation of Tibetan sheep[J].Jiangsu Journal of Agricultural Sciences,30(3):581-585.)
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何建文,刘秀,李少斌,等.2018.藏绵羊HMOX2基因克隆、表达及其耐低氧适应性进化分析[J].农业生物技术学报,26(11):1890-1899.(He J W,Liu X,Li S B,et al.2018.Cloning,expression and hypoxia adaptation evolution of HMOX2 gene in Tibetan sheep(Ovis aries)[J].Journal of Agricultural Biotechnology,26(11):1890-1899.)
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李瑶,王小明,吴其夏,等.2005.缺氧与血红素氧化酶-1的表达[J].国外医学,25(3):238-241.(Li Y,Wang X M,Wu Q Xet al.2005.Hypoxia and heme oxygenase-1 expression[J].Foreign Medical Science,25(3):238-241.)
李艳丽,徐功玉,肖金文,等.2017.血红素加氧酶1在斑马鱼低氧应激中的保护作用研究[J].水生生物学报,41(01)43-49.(Li Y L,Xu G Y,Xiao J W,et al.2017.Studies on the protective of zebrafish HO-1 in response to hypoxia[J].Acta Hydrobiolgica Sinica,41(01):43-49.)
马志明,张珍祥,韩泽民,等.2005.慢性阻塞性肺疾病患者血红素加氧酶-1基因启动子多态性对其基因表达的影响[J].新医学,36(8):444-446.(Ma Z M,Zhang Z XHan Z M,et al,2005.The effect of polymorphism of heme oxygenase-1 gene promoter on the expression of HO-1 gene in patients with chronic obstructive pulmonary disease[J].New Medicine,36(8):444-446.)
强巴央宗,张浩,白玛央宗,等.2011.高原环境中藏猪血液生理指标测定与比较[J].西南农业学报,24(6):2382-2384.(Chamba Y Z,Zhang H,Baima Y Z,et al.2011Determination of blood physiological parameters in Tibet pig at high altitude[J].Southwest China Journal of Agricultural Sciences,24(6):2382-2384.)
陶文庭,王琳琳,侯少丰,等.2014.斑马鱼HO1基因的表达特征及功能研究[J].水生生物学报,38(2):209-215(Tao W T,Wang L L,Hou S F,et al.2014.Study on expression pattern and function of zebrafish HO1[J].Acta Hydrobiolgica Sinica,38(2):209-215.)
信金伟,毛学荣,余忠祥,等.2007.欧拉型藏绵羊血红蛋白多态性研究[J].湖北农业科学,46(2):193-195.(Xin J WMao X R,Yu Z X,et al.2007.Research on polymorphism of hemoglobin in Oura Tibetam sheep[J].Hube Agricultural Sciences,46(2):193-195.)
赵雪,魏雪锋,连林生,等.2014.藏马低氧适应的血液生理指标研究[J].云南农业大学学报,29(5):684-688.(Zhao XWei X F,Lian L S,et al.2014.Study on blood physiological indicators of adaptation to hypoxia in Tibet Horse[J]Journal of Yunnan Agricultural University,29(5):684-688.)
Buis C I,Van der Steege G,Visser D S,et al.2008.Heme oxygenase-1 genotype of the donor is associated with graf survival after liver transplantation[J].American Journa of Transplantation,8(2):377-385.
Cruse I,Maines M D.Evidence suggesting that the two forms of heme oxygenase are products of different genes[J].Journal of Biological Chemistry,1988,263(7):3348-3353.
Ding X Z,Liang C N,Guo X,et al.2014.Physiological insigh into the high-altitude adaptations in domesticated yaks(Bos grunniens)along the Qinghai-Tibetan plateau altitudinal gradient[J].Livestock Science,162(2014):233-239.
GongH,ZhouHT,HickfordJG.2011.Diversityoftheglycine/tyrosine-rich keratin-associated protein 6 gene(KAP6)familyinsheep[J].MolecularBiologyReports,38(1):31-35.
Hachfi L,Simide R,Richard S,et al.2012.Effect of water temperature increase on HO-1 expression in European sea bass(Dicentrarchus labrax L.)tissues[J].Cellular and Molecular Biology,58(Suppl.):1752-1756.
Lakhal S,Schodel J,Townsend A R,et al.2011.Regulation of typeⅡtransmembrane serine proteinase TMPRSS6 by hypoxia-inducible factors:New link between hypoxia signaling and iron homeostasis[J].Journal of Biological Chemistry,286(6):4090-4097.
Lin Q,Weis S,Yang G,et al.2007.Heme Oxygenase-1 protein localizes to the nucleus and activates transcription factors important in oxidative stress[J].The Journal of Biological Chemistry,282(28):20621-20633.
Louise L D,Robyn G M,Jun N,et al.2014.New insights into intracellular locations and functionsof heme oxygenase-1[J].Antioxdants&Redoc Signalng,20(11):1723-1742.
Ryter S W,Alam J,Choi A M K.2006.Heme oxygenase-1/carbonmonoxide:From basic science to therapeutic applications[J].Physiological Reviews,86:583-650.
Storz J F,Scott G R,Cheviron Z A.2010.Phenotypic plasticity and genetic adaptation to high-altitude hypoxia in vertebrates[J].Journal of Experimental Biology,213(24):4125-4136.
Sun J L,Xia Z W,Zhang X H,et al.2003.The advances on the protective action and mechanism of heme oxygenase gene intissues and cells[J].Chinese Bulletin of Life Science,15(4):220-223.
Turkseven S,Kruger A,Mingone C J,et al.2005.The antioxdantmechanism ofheme oxygenase-l involves an increase in superoxide dismutase and catalase in experimental diabetes[J].American Journal of Physiology-heart and Circulatory Physiology,289(2):H701-707.
Wang D,Zhong X P,Qiao Z X,et al.2008.Inductive transcription and protective role of fish heme oxygenase-1 under hypoxic stress[J].Journal of Experimental Biology,211(16):2700-2706.
Zhang Q W,Zhang Y,Ma Y J,et al.2017.Molecular characteristics of the HO1 gene in yak are potentially adaptive for high altitude habitats[J].Journal of Computational and Theoretical Nanoscience,14(6):2698-2705.
Zheng J,Tian Q,Hou W,et al.2008.Tissue-specific expression of ALA synthase-1 and heme oxygenase-1 and their expression in livers of rats chronically exposed to ethanol[J].FEBS Letters,582(13):1829-1834.
成述儒,罗玉柱,韩建林,等.2009.利用微卫星DNA标记分析7个绵羊群体遗传多样性与系统发育[J].农业生物技术学报,17(5):808-814.(Cheng S R,Luo Y Z,Han J L,et al.2009.Analysis of genetic diversity and phylogeny among 7sheep population using microsatellite DNA marker[J].Journal of Agricultural Biotechnology,17(5):808-814.)
何建文,刘秀,李少斌,等.2018.藏绵羊HMOX2基因克隆、表达及其耐低氧适应性进化分析[J].农业生物技术学报,26(11):1890-1899.(He J W,Liu X,Li S B,et al.2018.Cloning,expression and hypoxia adaptation evolution of HMOX2 gene in Tibetan sheep(Ovis aries)[J].Journal of Agricultural Biotechnology,26(11):1890-1899.)
林杰,景孟环,张越红,等.2006.低氧预处理对心脏成纤维细胞血红素氧化酶1基因表达的影响[J].中国医科大学学报,35(4):377-379.(Lin J,Jing M H,Zhang Y H,et al.2006.Expressionof hemeoxygenase-1 mRNA after hypoxic preconditioning in neonatal ratcardiac fibroblasts[J].Journal of China Medical University,35(4):377-379.)
李瑶,王小明,吴其夏,等.2005.缺氧与血红素氧化酶-1的表达[J].国外医学,25(3):238-241.(Li Y,Wang X M,Wu Q Xet al.2005.Hypoxia and heme oxygenase-1 expression[J].Foreign Medical Science,25(3):238-241.)
李艳丽,徐功玉,肖金文,等.2017.血红素加氧酶1在斑马鱼低氧应激中的保护作用研究[J].水生生物学报,41(01)43-49.(Li Y L,Xu G Y,Xiao J W,et al.2017.Studies on the protective of zebrafish HO-1 in response to hypoxia[J].Acta Hydrobiolgica Sinica,41(01):43-49.)
马志明,张珍祥,韩泽民,等.2005.慢性阻塞性肺疾病患者血红素加氧酶-1基因启动子多态性对其基因表达的影响[J].新医学,36(8):444-446.(Ma Z M,Zhang Z XHan Z M,et al,2005.The effect of polymorphism of heme oxygenase-1 gene promoter on the expression of HO-1 gene in patients with chronic obstructive pulmonary disease[J].New Medicine,36(8):444-446.)
强巴央宗,张浩,白玛央宗,等.2011.高原环境中藏猪血液生理指标测定与比较[J].西南农业学报,24(6):2382-2384.(Chamba Y Z,Zhang H,Baima Y Z,et al.2011Determination of blood physiological parameters in Tibet pig at high altitude[J].Southwest China Journal of Agricultural Sciences,24(6):2382-2384.)
陶文庭,王琳琳,侯少丰,等.2014.斑马鱼HO1基因的表达特征及功能研究[J].水生生物学报,38(2):209-215(Tao W T,Wang L L,Hou S F,et al.2014.Study on expression pattern and function of zebrafish HO1[J].Acta Hydrobiolgica Sinica,38(2):209-215.)
信金伟,毛学荣,余忠祥,等.2007.欧拉型藏绵羊血红蛋白多态性研究[J].湖北农业科学,46(2):193-195.(Xin J WMao X R,Yu Z X,et al.2007.Research on polymorphism of hemoglobin in Oura Tibetam sheep[J].Hube Agricultural Sciences,46(2):193-195.)
赵雪,魏雪锋,连林生,等.2014.藏马低氧适应的血液生理指标研究[J].云南农业大学学报,29(5):684-688.(Zhao XWei X F,Lian L S,et al.2014.Study on blood physiological indicators of adaptation to hypoxia in Tibet Horse[J]Journal of Yunnan Agricultural University,29(5):684-688.)
Buis C I,Van der Steege G,Visser D S,et al.2008.Heme oxygenase-1 genotype of the donor is associated with graf survival after liver transplantation[J].American Journa of Transplantation,8(2):377-385.
Cruse I,Maines M D.Evidence suggesting that the two forms of heme oxygenase are products of different genes[J].Journal of Biological Chemistry,1988,263(7):3348-3353.
Ding X Z,Liang C N,Guo X,et al.2014.Physiological insigh into the high-altitude adaptations in domesticated yaks(Bos grunniens)along the Qinghai-Tibetan plateau altitudinal gradient[J].Livestock Science,162(2014):233-239.
GongH,ZhouHT,HickfordJG.2011.Diversityoftheglycine/tyrosine-rich keratin-associated protein 6 gene(KAP6)familyinsheep[J].MolecularBiologyReports,38(1):31-35.
Hachfi L,Simide R,Richard S,et al.2012.Effect of water temperature increase on HO-1 expression in European sea bass(Dicentrarchus labrax L.)tissues[J].Cellular and Molecular Biology,58(Suppl.):1752-1756.
Lakhal S,Schodel J,Townsend A R,et al.2011.Regulation of typeⅡtransmembrane serine proteinase TMPRSS6 by hypoxia-inducible factors:New link between hypoxia signaling and iron homeostasis[J].Journal of Biological Chemistry,286(6):4090-4097.
Lin Q,Weis S,Yang G,et al.2007.Heme Oxygenase-1 protein localizes to the nucleus and activates transcription factors important in oxidative stress[J].The Journal of Biological Chemistry,282(28):20621-20633.
Louise L D,Robyn G M,Jun N,et al.2014.New insights into intracellular locations and functionsof heme oxygenase-1[J].Antioxdants&Redoc Signalng,20(11):1723-1742.
Ryter S W,Alam J,Choi A M K.2006.Heme oxygenase-1/carbonmonoxide:From basic science to therapeutic applications[J].Physiological Reviews,86:583-650.
Storz J F,Scott G R,Cheviron Z A.2010.Phenotypic plasticity and genetic adaptation to high-altitude hypoxia in vertebrates[J].Journal of Experimental Biology,213(24):4125-4136.
Sun J L,Xia Z W,Zhang X H,et al.2003.The advances on the protective action and mechanism of heme oxygenase gene intissues and cells[J].Chinese Bulletin of Life Science,15(4):220-223.
Turkseven S,Kruger A,Mingone C J,et al.2005.The antioxdantmechanism ofheme oxygenase-l involves an increase in superoxide dismutase and catalase in experimental diabetes[J].American Journal of Physiology-heart and Circulatory Physiology,289(2):H701-707.
Wang D,Zhong X P,Qiao Z X,et al.2008.Inductive transcription and protective role of fish heme oxygenase-1 under hypoxic stress[J].Journal of Experimental Biology,211(16):2700-2706.
Zhang Q W,Zhang Y,Ma Y J,et al.2017.Molecular characteristics of the HO1 gene in yak are potentially adaptive for high altitude habitats[J].Journal of Computational and Theoretical Nanoscience,14(6):2698-2705.
Zheng J,Tian Q,Hou W,et al.2008.Tissue-specific expression of ALA synthase-1 and heme oxygenase-1 and their expression in livers of rats chronically exposed to ethanol[J].FEBS Letters,582(13):1829-1834.
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