猪CRHR1、CRHR2基因的克隆及mRNA表达规律的研究
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摘要
在现代化养猪生产中,存在着断奶应激、屠宰应激、热应激等诸多应激因素。这些因素严重影响着养猪业的发展。对此,人们已从饲养管理方面提出了许多对策,但对猪应激的遗传机制、分子机理及从遗传角度进行改良的研究却很少。
应激产生时,下丘脑-垂体-肾上腺(HPA)轴兴奋,下丘脑释放促肾上腺皮质激素释放激素(CRH)以及神经肽激素到下丘脑门脉系统与位于脑垂体前部的促皮质激素细胞中的促肾上腺皮质激素释放激素受体(CRHR)结合,并激发促肾上腺皮质激素(ACTH)的分泌。ACTH通过血液作用于外周器官肾上腺皮质,使其释放糖皮质激素(GC),糖皮质激素能够影响整个机体,是机体对应激适应的关键激素。
本文以猪为研究对象,对CRH受体基因CRHR1、CRHR2进行克隆、核酸序列分析、蛋白质结构功能预测,并探讨了这两个基因的mRNA表达规律,目的在于从应激的生理过程出发研究猪应激的分子机理,从遗传上找到猪抗应激的有效方法,进而推动猪的抗病育种,同时为以猪为动物模型进行人类疾病的研究提供理论依据。
本研究的具体内容及结论如下:
1、利用同源克隆与电子克隆结合的方法首次从猪脑组织中克隆得到CRHR1、CRHR2基因的全长编码序列(CDS)。CRHR1基因包含一个长为1248bp的开放阅读框,编码415个氨基酸。CRHR2基因包含一个长为1236bp的开放阅读框,编码411个氨基酸。结合已有的数据库对序列进行分析,发现了6个单核苷酸多态性(SNP)位点。
2、将猪CRHR1和CRHR2基因与人、大鼠、小鼠、牛、狗等物种同源基因的CDS进行序列比对,表明CRHR1基因与其它哺乳动物的CDS相似性达91%以上,CRHR2基因与其它哺乳动物的CDS相似性达86%以上。猪CRHR1和CRHR2氨基酸序列与其它哺乳动物同源的氨基酸序列比较,相似性分别达97%和88%以上。这些结果说明CRHR1和CRHR2基因在哺乳动物中是高度保守的。
3、应用生物信息学的方法对猪CRHR1和CRHR2蛋白质的基本特性、结构和功能进行预测。结果表明猪CRHR1和CRHR2同属于G蛋白偶联受体Ⅱ家族,属于类分泌素蛋白,包含一个GPCR结构域,包括4个胞外域、7个跨膜域和4个胞内域。CRHR1和CRHR2蛋白质结构还包含一个激素受体结构域,表明它可能具有激素受体的特征和基本功能。生物信息学的结果表明在对蛋白的结构和功能起关键作用的区域上,猪CRHR1和CRHR2蛋白是保守的。说明猪CRHR1和CRHR2蛋白具有与已知功能蛋白相似的功能。
4、利用实时荧光定量PCR方法对21日龄断奶仔猪与未断奶仔猪脑中CRHR1和CRHR2基因mRNA表达量进行检测,结果表明断奶应激对采集的仔猪脑中CRHR1和CRHR2基因mRNA表达量表现出一定的差异,但未达到显著水平(p>0.05)。
In the modern swine production, the stress syndrome, including weaning stress, the slaughter stress, heat stress and so on, brings great loss in the pig industry economically. As a result, a great deal of efforts has been made to control porcine stress, especially on the sides of feed and environment management. But pig stress is controlled by not only environmental but also genetic factors. Thus, it is indispensable to uncover the molecular genetic mechanism of the pig stress.
As stress arises, the excitement of hypothalamus-pituitary-adrenal (HPA) axis causes the release of hypothalamic corticotropin-releasing hormone (CRH), as well as neuropeptide hormone to the hypothalamic portal system, which stimulates adrenocorticotropic hormone (ACTH) secretion by binding the corticotropin-releasing hormone receptor (CRHR) in the cell in the front of pituitary. With the effect of ACTH in blood on the adrenal cortex in the peripheral organs, glucocorticoid (GC) is released. Finally, GC is a key hormone to the stress which can affect the whole body.
Therefore, in order to uncover the molecular mechanism of pig stress, this study focused on pig CRH receptor genes CRHR1 and CRHR2 basing on the physiological process of pig stress described above. Gene cloning, sequence analysis, protein structure and function prediction of these two genes were finished as well as their mRNA expression analysis in the brain of weaning piglets.
The main research works and conclusions are as follows:
(1) The first complete coding sequence (CDS) of CRHR1 and CRHR2 are obtained using the approach of combining homology and electronic cloning. CRHR1 gene contains a 1,248 bp open reading frame (ORF) encoding 415 amino acids while CRHR2 gene contains a 1,236 bp ORF encoding 411 amino acids. Analysing the two genes and database, we found six single nucleotide polymorphism sites.
(2) The alignment analysis of CDS sequences of CRHR1 and CRHR2 among rats, mice, cows, dogs and pigs shows that the consistency of CRHR1 gene in pig and other mammals is more than 91%, while the comparability of CRHR2 is more than 86%. The comparability of the protein sequences of CRHR1 and CRHR2 of pig with other mammals are more than 97% and 88% respectively. It verified that CRHR1 and CRHR2 gene are highly conserved in mammals.
(3) We predicted the basic features, protein structure and molecular function of porcine CRHR1 and CRHR2 proteins with bioinformatics methods. The results indicate that porcine CRHR1 and CRHR2 belong to G-protein-coupled receptor family, which consist of a GPCR domain, including four extracellular domains, seven transmembrane domains and four intracellular domains. CRHR1 and CRHR2 proteins structure also contains a hormone receptor domain that suggests those two genes have the basic characteristics and functions of hormone receptor. Bioinformatics results show that porcine CRHR1 and CRHR2 proteins are very conservative in the key structural and functional region. This suggests that porcine CRHR1 and CRHR2 proteins should have the similar function with some known proteins.
(4) CRHR1 and CRHR2 mRNA expression was detected in the brains of weaning and non-weaning piglets using real-time quantitative PCR method. The result shows the effects of weaning stress on the mRNA expression level of CRHR1 and CRHR2 in the piglet brain exist though not significant (p>0.05) statistically.
Obviously, our research built an important foundation for finding the effective genetic anti-stress approach and developing the anti-disease breeding of pig.
应激产生时,下丘脑-垂体-肾上腺(HPA)轴兴奋,下丘脑释放促肾上腺皮质激素释放激素(CRH)以及神经肽激素到下丘脑门脉系统与位于脑垂体前部的促皮质激素细胞中的促肾上腺皮质激素释放激素受体(CRHR)结合,并激发促肾上腺皮质激素(ACTH)的分泌。ACTH通过血液作用于外周器官肾上腺皮质,使其释放糖皮质激素(GC),糖皮质激素能够影响整个机体,是机体对应激适应的关键激素。
本文以猪为研究对象,对CRH受体基因CRHR1、CRHR2进行克隆、核酸序列分析、蛋白质结构功能预测,并探讨了这两个基因的mRNA表达规律,目的在于从应激的生理过程出发研究猪应激的分子机理,从遗传上找到猪抗应激的有效方法,进而推动猪的抗病育种,同时为以猪为动物模型进行人类疾病的研究提供理论依据。
本研究的具体内容及结论如下:
1、利用同源克隆与电子克隆结合的方法首次从猪脑组织中克隆得到CRHR1、CRHR2基因的全长编码序列(CDS)。CRHR1基因包含一个长为1248bp的开放阅读框,编码415个氨基酸。CRHR2基因包含一个长为1236bp的开放阅读框,编码411个氨基酸。结合已有的数据库对序列进行分析,发现了6个单核苷酸多态性(SNP)位点。
2、将猪CRHR1和CRHR2基因与人、大鼠、小鼠、牛、狗等物种同源基因的CDS进行序列比对,表明CRHR1基因与其它哺乳动物的CDS相似性达91%以上,CRHR2基因与其它哺乳动物的CDS相似性达86%以上。猪CRHR1和CRHR2氨基酸序列与其它哺乳动物同源的氨基酸序列比较,相似性分别达97%和88%以上。这些结果说明CRHR1和CRHR2基因在哺乳动物中是高度保守的。
3、应用生物信息学的方法对猪CRHR1和CRHR2蛋白质的基本特性、结构和功能进行预测。结果表明猪CRHR1和CRHR2同属于G蛋白偶联受体Ⅱ家族,属于类分泌素蛋白,包含一个GPCR结构域,包括4个胞外域、7个跨膜域和4个胞内域。CRHR1和CRHR2蛋白质结构还包含一个激素受体结构域,表明它可能具有激素受体的特征和基本功能。生物信息学的结果表明在对蛋白的结构和功能起关键作用的区域上,猪CRHR1和CRHR2蛋白是保守的。说明猪CRHR1和CRHR2蛋白具有与已知功能蛋白相似的功能。
4、利用实时荧光定量PCR方法对21日龄断奶仔猪与未断奶仔猪脑中CRHR1和CRHR2基因mRNA表达量进行检测,结果表明断奶应激对采集的仔猪脑中CRHR1和CRHR2基因mRNA表达量表现出一定的差异,但未达到显著水平(p>0.05)。
In the modern swine production, the stress syndrome, including weaning stress, the slaughter stress, heat stress and so on, brings great loss in the pig industry economically. As a result, a great deal of efforts has been made to control porcine stress, especially on the sides of feed and environment management. But pig stress is controlled by not only environmental but also genetic factors. Thus, it is indispensable to uncover the molecular genetic mechanism of the pig stress.
As stress arises, the excitement of hypothalamus-pituitary-adrenal (HPA) axis causes the release of hypothalamic corticotropin-releasing hormone (CRH), as well as neuropeptide hormone to the hypothalamic portal system, which stimulates adrenocorticotropic hormone (ACTH) secretion by binding the corticotropin-releasing hormone receptor (CRHR) in the cell in the front of pituitary. With the effect of ACTH in blood on the adrenal cortex in the peripheral organs, glucocorticoid (GC) is released. Finally, GC is a key hormone to the stress which can affect the whole body.
Therefore, in order to uncover the molecular mechanism of pig stress, this study focused on pig CRH receptor genes CRHR1 and CRHR2 basing on the physiological process of pig stress described above. Gene cloning, sequence analysis, protein structure and function prediction of these two genes were finished as well as their mRNA expression analysis in the brain of weaning piglets.
The main research works and conclusions are as follows:
(1) The first complete coding sequence (CDS) of CRHR1 and CRHR2 are obtained using the approach of combining homology and electronic cloning. CRHR1 gene contains a 1,248 bp open reading frame (ORF) encoding 415 amino acids while CRHR2 gene contains a 1,236 bp ORF encoding 411 amino acids. Analysing the two genes and database, we found six single nucleotide polymorphism sites.
(2) The alignment analysis of CDS sequences of CRHR1 and CRHR2 among rats, mice, cows, dogs and pigs shows that the consistency of CRHR1 gene in pig and other mammals is more than 91%, while the comparability of CRHR2 is more than 86%. The comparability of the protein sequences of CRHR1 and CRHR2 of pig with other mammals are more than 97% and 88% respectively. It verified that CRHR1 and CRHR2 gene are highly conserved in mammals.
(3) We predicted the basic features, protein structure and molecular function of porcine CRHR1 and CRHR2 proteins with bioinformatics methods. The results indicate that porcine CRHR1 and CRHR2 belong to G-protein-coupled receptor family, which consist of a GPCR domain, including four extracellular domains, seven transmembrane domains and four intracellular domains. CRHR1 and CRHR2 proteins structure also contains a hormone receptor domain that suggests those two genes have the basic characteristics and functions of hormone receptor. Bioinformatics results show that porcine CRHR1 and CRHR2 proteins are very conservative in the key structural and functional region. This suggests that porcine CRHR1 and CRHR2 proteins should have the similar function with some known proteins.
(4) CRHR1 and CRHR2 mRNA expression was detected in the brains of weaning and non-weaning piglets using real-time quantitative PCR method. The result shows the effects of weaning stress on the mRNA expression level of CRHR1 and CRHR2 in the piglet brain exist though not significant (p>0.05) statistically.
Obviously, our research built an important foundation for finding the effective genetic anti-stress approach and developing the anti-disease breeding of pig.
引文
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