牦牛HYOU1基因克隆及组织表达分析
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摘要
为探究HYOU1基因在牦牛中的组织表达谱,通过对西藏牦牛HYOU1基因的CDS区进行克隆测序,分析该基因的结构和功能,采用RT-PCR技术检测黄牛和牦牛肺脏、心脏、肝脏、乳腺、大脑及肌肉中HYOU1基因的相对表达量。结果表明:(1)HYOU1基因CDS区长度为3 006 bp,其中A、G、C和T含量分别为25.0%、31.1%、26.7%和17.1%,存在一定碱基偏好性;发现1个(ACG→ACA)SNP位点,为同义突变。(2)生物信息学分析表明该蛋白呈中性,为较不稳定、亲水性分泌信号蛋白;存在一个跨膜螺旋(TMhelix)区位于13-35氨基酸位置;二、三级蛋白结构分析发现其主要的空间构象为:无规则卷曲及α-螺旋。(3)聚类分析显示,西藏牦牛与野牦牛的遗传距离最近,与瘤牛及普通牛的遗传距离次之,与水牛最远。(4)RT-PCR结果显示HYOU1基因在黄牛和牦牛肝脏、乳腺、肺脏、大脑、心脏、肌肉6个组织中均有表达,且相对表达量依次递减;在相同组织中,黄牛组织的表达量均显著高于牦牛组织中的表达量(P<0.05),其中黄牛肝脏组织的表达量为牦牛表达量的4.4倍。
In order to understand the tissue expression profile of gene HYOU1,the CDS region of HYOU1 in Tibetan yak was cloned and sequenced,and the structure and function of gene HYOU1 were analyzed;furthermore,the mRNA expression profile of gene HYOU1 in the lung,heart,liver,mammary gland,brain and muscle of cattle and yak were detected by RT-PCR. The results were as followings.(1)The CDS region of gene HYOU1 was 3 006 bp,with the percentage of A,G,C and T being 25.0%,31.1%,26.7% and 17.1%,respectively,and there was base preference. One SNP site(ACG → ACA)was identified as a synonymous mutation.(2)Bioinformatics analysis showed that the protein was a neutral,relatively unstable and hydrophilic secretion signal one,and a transmembrane helix(TMhelix)region were obtained and located at the 13-35 amino acid sites. The second and tertiary structures analysis showed that the main spatial structure was random coil and α-helix.(3)Cluster analysis demonstrated that the gene HYOU1 of the Tibetan yak was the closest associated with wild yak,followed by zebu and cattle,and the most distant with buffalo.(4)RT-PCR results indicated that gene HYOU1 expressed in 6 tissues liver,mammary gland,lung,brain,heart,and muscle of cattle and yak at descending order. In addition,for the same tissue,the expression level of the cattle was significantly higher than that of the yak(P<0.05),i.e.,the expression in the liver of the cattle was 4.4 times higher than that of yak.
In order to understand the tissue expression profile of gene HYOU1,the CDS region of HYOU1 in Tibetan yak was cloned and sequenced,and the structure and function of gene HYOU1 were analyzed;furthermore,the mRNA expression profile of gene HYOU1 in the lung,heart,liver,mammary gland,brain and muscle of cattle and yak were detected by RT-PCR. The results were as followings.(1)The CDS region of gene HYOU1 was 3 006 bp,with the percentage of A,G,C and T being 25.0%,31.1%,26.7% and 17.1%,respectively,and there was base preference. One SNP site(ACG → ACA)was identified as a synonymous mutation.(2)Bioinformatics analysis showed that the protein was a neutral,relatively unstable and hydrophilic secretion signal one,and a transmembrane helix(TMhelix)region were obtained and located at the 13-35 amino acid sites. The second and tertiary structures analysis showed that the main spatial structure was random coil and α-helix.(3)Cluster analysis demonstrated that the gene HYOU1 of the Tibetan yak was the closest associated with wild yak,followed by zebu and cattle,and the most distant with buffalo.(4)RT-PCR results indicated that gene HYOU1 expressed in 6 tissues liver,mammary gland,lung,brain,heart,and muscle of cattle and yak at descending order. In addition,for the same tissue,the expression level of the cattle was significantly higher than that of the yak(P<0.05),i.e.,the expression in the liver of the cattle was 4.4 times higher than that of yak.
引文
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[8]Jung TW, Lee KT, Lee MW, et al. SIRT1 attenuates palmitateinduced endoplasmic reticulum stress and insulin resistance in HepG2 cells via induction of oxygen-regulated protein 150[J].Biochemical&Biophysical Research Communications, 2012, 422(2):229-232.
[9]汪琦.牦牛四个低氧适应基因的遗传多态性研究[D].成都:西南民族大学, 2017.
[10]Tamatani M, Matsuyama T, Yamaguchi A, et al. ORP150 protects against hypoxia/ischemia-induced neuronal death.[J]. Nature Medicine, 2001, 7(3):317-323.
[11]Ozawa K, Kuwabara K, Tamatani M, et al. 150-kDa oxygenregulated protein(ORP150)suppresses hypoxia-induced apoptotic cell death.[J]. Journal of Biological Chemistry, 1999,274(10):6397-6404.
[12]Liu Y, Wang XC, Hu D, et al. Heat shock protein 70 protects PC12 cells against ischemia-hypoxia/reoxygenation by maintaining intracellular Ca2+homeostasis[J]. Neural Regeneration Research,2016, 11(7):1134-1140.
[13]Wei J, Tong J, Yu L, et al. EMF protects cardiomyocytes against hypoxia-induced injury via heat shock protein 70 activation.[J].Chemico-Biological Interactions, 2016, 25(3):8-17.
[14]Montesi M, J?hn K, Bonewald L, et al. Hypoxia mediates osteocyte ORP150 expression and cell death in vitro[J]. Molecular Medicine Reports, 2016, 14(5):4248-4254.
[15]Kr?towski R, Borzym-Kluczyk M, Cechowska-Pasko M. Hypoxia enhances the senescence effect of bortezomib-the proteasome inhibitor-on human skin fibroblasts[J]. Biomed Research International, 2014, 2014:196249.
[16]Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))method[J]. Methods, 2001, 25(4):402-408.
[17]景炅婕,张静,贾夏丽,等.绵羊及相关物种去乙酰化酶1的理化性质和蛋白结构预测[J].山西农业大学学报:自然科学版, 2015, 35(2):124-132.
[18]宋大伟.牦牛的起源与系统发育分析[D].南京:南京农业大学, 2008.
[19]赵成玉,圈启芳,何熠伟,等.慢性持续性低氧对大鼠肝脏和胰腺GLUT2 m RNA表达的影响[J/OL].中华临床医师杂志,2015(22):4148-4152.
[20]龚俊艳,赵成玉.持续性低氧对糖代谢的调节机制的研究进展[J].中国全科医学, 2018, 21(12):1497-1503.
[21]龚俊艳,赵成玉.持续性低氧大鼠肝脏HIF-2α信号通路变化及对血糖的影响[J].中国高原医学与生物学杂志, 2018, 39(2):89-94.
[22]韩树鑫,苟潇,杨舒黎.动物低氧适应的生理与分子机制[J].中国畜牧兽医, 2010, 37(9):29-34.
[2]Wang Z, Yonezawa T, Liu B, et al. Domestication Relaxed selective constraints on the yak mitochondrial genome[J]. Molecular Biology&Evolution, 2011, 28(5):1553-1556.
[3]钟金城.牦牛遗传与育种[M].成都:四川科学技术出版社,1996.
[4]国家畜禽遗传资源委员会.中国畜禽遗传资源志:牛志[M].北京:中国农业出版社, 2011.
[5]董传豪,饶开晴,徐亚欧,等.高原动物的低氧适应性研究进展[J].黑龙江畜牧兽医, 2015(17):57-60.
[6]Ikeda J, Kaneda S, Kuwabara K, et al. Cloning and expression ofcDNAencodingthehuman150kDaoxygen-regulated protein, ORP150[J]. Biochemical&Biophysical Research Communications, 1997, 230(1):94-99.
[7]Kuwabara K, Matsumoto M, Ikeda J, et al. Purification and characterization of a novel stress protein, the 150-kDa oxygenregulated protein(ORP150), from cultured rat astrocytes and its expression in ischemic mouse brain[J]. Journal of Biological Chemistry, 1996, 25(9):5025-5032.
[8]Jung TW, Lee KT, Lee MW, et al. SIRT1 attenuates palmitateinduced endoplasmic reticulum stress and insulin resistance in HepG2 cells via induction of oxygen-regulated protein 150[J].Biochemical&Biophysical Research Communications, 2012, 422(2):229-232.
[9]汪琦.牦牛四个低氧适应基因的遗传多态性研究[D].成都:西南民族大学, 2017.
[10]Tamatani M, Matsuyama T, Yamaguchi A, et al. ORP150 protects against hypoxia/ischemia-induced neuronal death.[J]. Nature Medicine, 2001, 7(3):317-323.
[11]Ozawa K, Kuwabara K, Tamatani M, et al. 150-kDa oxygenregulated protein(ORP150)suppresses hypoxia-induced apoptotic cell death.[J]. Journal of Biological Chemistry, 1999,274(10):6397-6404.
[12]Liu Y, Wang XC, Hu D, et al. Heat shock protein 70 protects PC12 cells against ischemia-hypoxia/reoxygenation by maintaining intracellular Ca2+homeostasis[J]. Neural Regeneration Research,2016, 11(7):1134-1140.
[13]Wei J, Tong J, Yu L, et al. EMF protects cardiomyocytes against hypoxia-induced injury via heat shock protein 70 activation.[J].Chemico-Biological Interactions, 2016, 25(3):8-17.
[14]Montesi M, J?hn K, Bonewald L, et al. Hypoxia mediates osteocyte ORP150 expression and cell death in vitro[J]. Molecular Medicine Reports, 2016, 14(5):4248-4254.
[15]Kr?towski R, Borzym-Kluczyk M, Cechowska-Pasko M. Hypoxia enhances the senescence effect of bortezomib-the proteasome inhibitor-on human skin fibroblasts[J]. Biomed Research International, 2014, 2014:196249.
[16]Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))method[J]. Methods, 2001, 25(4):402-408.
[17]景炅婕,张静,贾夏丽,等.绵羊及相关物种去乙酰化酶1的理化性质和蛋白结构预测[J].山西农业大学学报:自然科学版, 2015, 35(2):124-132.
[18]宋大伟.牦牛的起源与系统发育分析[D].南京:南京农业大学, 2008.
[19]赵成玉,圈启芳,何熠伟,等.慢性持续性低氧对大鼠肝脏和胰腺GLUT2 m RNA表达的影响[J/OL].中华临床医师杂志,2015(22):4148-4152.
[20]龚俊艳,赵成玉.持续性低氧对糖代谢的调节机制的研究进展[J].中国全科医学, 2018, 21(12):1497-1503.
[21]龚俊艳,赵成玉.持续性低氧大鼠肝脏HIF-2α信号通路变化及对血糖的影响[J].中国高原医学与生物学杂志, 2018, 39(2):89-94.
[22]韩树鑫,苟潇,杨舒黎.动物低氧适应的生理与分子机制[J].中国畜牧兽医, 2010, 37(9):29-34.