两个贵州山羊品种GHSR和GHRL基因遗传变异及其与生长性状的关联性

doi:10.3864/j.issn.0578-1752.2015.01.14

摘要/Abstract

摘要： 【目的】探讨GHSR和GHRL基因作为山羊体重体尺性状候选基因的可能性，寻找与山羊生长性状相关的分子遗传标记。【方法】以贵州白山羊和贵州黑山羊为研究素材，采用DNA混合池结合PCR产物直接测序及PCR-SSCP技术检测GHSR和GHRL基因的单核苷酸多态性，利用SPSS (18.0)软件GLM统计模型分析基因SNPs不同基因型及合并基因型与贵州山羊生长性状的关联性，同时采用在线软件对GHSR基因进行生物信息学分析。【结果】在GHSR基因外显子2和3′UTR分别检测到G200A同义突变和T628C位点，而在5′UTR区和外显子1则未发现多态性。设计一对特异性引物对G200A位点进行PCR-SSCP分析，表现为3种基因型，依次命名为GG、GA和AA。GG基因型为优势基因型，在贵州白山羊和贵州黑山羊母羊群体中等位基因G为优势等位基因，其等位基因频率分别为0.6731和0.5243。而在贵州黑山羊公羊群体中A则为优势等位基因，其频率为0.5122。多态信息含量均表现为中度多态(0.25＜PIC＜0.50)。在GHRL基因内含子2处发现1个G141A突变，外显子2和外显子4处分别检测到2个同义突变(T78C和C14T)，设计一对特异性引物对C14T位点进行SSCP分析，显示为2种基因型CT和CC。在所有试验群体中，CC基因型为优势基因型，其频率分别为0.7692、0.9417和0.9390，等位基因C为优势等位基因，其频率依次为0.8846、0.9709和0.9695，多态信息含量均显示为低度多态(PIC＜0.25)。χ²检验显示所有试验群体G200A和C14T位点基因型分布均符合Hardy-Weinberg平衡(P＞0.05)。关联分析表明，GHSR基因G200A位点与山羊体重、体高、体斜长和管围显著相关 (P＜0.05)，纯合子GG基因型优势较为明显，贵州白山羊GG基因型个体的体高显著高于GA基因型个体和管围显著高于AA基因型个体(P＜0.05)。C14T位点贵州黑山羊 (公羊) CC基因型个体的体重、体高和胸围显著高于CT基因型 (P＜0.05)。组合基因型对体高和胸围指标的影响显著(P＜0.05)，表现为CCGG合并基因型个体的体高显著高于CCAA合并基因型个体 (P＜0.05)。生物信息学分析揭露，GHSR基因5’UTR处未检测到核心启动子区和CpG岛，仅发现1个CAAT-box和部分潜在的转录因子结合位点，G200A导致mRNA二级结构发生明显变化。GHSR蛋白二级结构以α-螺旋为主 (48.90%)，三级结构及跨膜螺旋结构预测表明该蛋白是膜蛋白。【结论】研究初步推测GHSR基因和GHRL基因多态性及合并基因型对山羊生长性状具有较显著的影响，G200A和C14T位点可作为山羊生长性状的有效遗传标记用于指导山羊的分子育种。

关键词: GHSR基因, GHRL基因, 遗传变异, 生长性状, 关联分析

Abstract: 【Objective】 The aim of this study was to explore the relationships between GHSR and GHRL gene and body weight & size traits, and search the molecular genetic markers related to goat growth traits.【Method】Guizhou white goat and Guizhou black goat were chosen as subjects, the DNA pooling was constructed and the technology of direct sequencing of PCR products and PCR-SSCP were used to detect the single nucleotide polymorphism of GHSR and GHRL genes, and its association of polymorphisms and different genotypes and combined genotypes with growth traits was analyzed by SPSS(18.0). Meanwhile, the bioinformatics analysis of GHSR gene was conducted by online software.【Result】The result showed that two SNPs (G200A and T628C) were detected in exon 2 and 3′UTR but no SNPs in exon1 and 5′UTR of GHSR gene, respectively. G200A was a sense mutation site and devided into three genotypes GG, GA and AA by PCR-SSCP. Genotype GG was the dominant genotype and allele G was dominant allele with the frequencies of 0.6731 and 0.5243 in Guizhou white goat and Guizhou black goat (♀) groups instead of allele A in Guizhou black goat (♂). All populations were moderate polymorphic in G200A site (0.25＜PIC＜0.50). G141A was found in intron 2 and two sense mutation sites (T78C and C14T) were identified in exon2 and exon4 of GHRL gene, respectively. There were two genotypes (CT and CC) in C14T site, genotype CC was the dominant genotype, which the frequency were 0.7692, 0.9417 and 0.9390, allele C was dominant allele with the frequency of 0.8846, 0.9709 and 0.9695, respectively. These populations were low polymorphic (PIC＜0.25). χ²test indicated that SNPs(G200A and C14T) fit with Hardy-Weinberg equilibrium in the population (P＞0.05). Correlation analysis revealed that G200A site was significantly associated with body weight, body height, body length and hucklebone width (P＜0.05), GG genotype was superior genotype. Additionally, body weight, body height and chest girth of individuals of Guizhou black goat (♂)with genotype CC was significantly higher than those with genotype CT(P＜0.05) in C14T site. Meanwhile, the combined genotype was significantly associated with body height and chest girth, the individuals with CCGG genotype had significantly better body height than that of genotype CCAA (P＜0.05). Bioinformatics analysis indicated that no core promoter regions and CpG island were found but a CAAT-box and several important transcription factors in 5′UTR region of GHSR gene. G200A had led to the change of secondary structure of mRNA. The secondary structure of protein was mainly α-helix (48.90%). Tertiary structure and transmembrane helix prediction showed that GHSR protein was a membrane protein.【Conclusion】The results revealed that SNPs and combined genotype of GHSR and GHRL gene had effect on growth traits in goat, G200A and C14T sites could be used as effective genetic markers for goat molecular breeding.

Key words: GHSR gene, GHRL gene, genetic variation, growth traits, correlation analysis

宋桃伟，蔡惠芬，罗卫星，刘若余，张依裕，孙岩岩，刘彬. 两个贵州山羊品种GHSR和GHRL基因遗传变异及其与生长性状的关联性[J]. 中国农业科学, 2015, 48(1): 140-153.

SONG Tao-wei, CAI Hui-fen, LUO Wei-xing, LIU Ruo-yu, ZHANG Yi-yu, SUN Yan-yan, LIU Bin. Association of GHSR and GHRL Gene Genetic Variation with Growth Traits in Two Guizhou Goat Breeds[J]. Scientia Agricultura Sinica, 2015, 48(1): 140-153.

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参考文献

[1] Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, Matsukura S. A role for ghrelin in the central regulation of feeding. Nature, 2001, 409:194-198.

[2] Arvat E, Di-Vito, LBroglio F, Papotti, M, Muccioli G, Dieguez C, Casanueva F F, Deghenghi R, Camanni F, Ghigo E. Preliminary evidence that Ghrelin, the natural GH secretagogue (GHS)-receptor ligand, strongly stimulates GH secretion in humans. Journal of Endocrinological Investigation, 2000, 23(8):493-495.

[3] Sun Y, Butte N F, Garcia J M, Smith R G. Characterization of adult ghrelin and ghrelin receptor knockout mice under positive and negative energy balance. Endocrinology, 2008, 149(2):843-850.

[4] Nie Q, Fang M, Xie L, Peng X, Xu H, Luo C, Zhang D, Zhang X. Molecular characterization of the ghrelin and ghrelin receptor genes and effects on fat deposition in chicken and duck. Journal of Biomedicine and Biotechnology, 2009, ID: 567120.

[5] 邱文才, 王维刚. 生长激素促分泌素受体及其内源性配体ghrelin与胃肠运动. 中国普通外科杂志, 2007, 16(10): 1007-1009.

Qiu W C, Wang W G. Study on the growth hormone secretagogue receptor and its endogenous ligand (ghrelin) and gastrointestinal motility. Chinese Journal of General Surgery, 2007, 16(10): 1007-1009. (in Chinese).

[6] Hosoda H, Kojima M, Kangawa, K. Biological, physiological and pharmacological aspects of ghrelin. Journal of Pharmacological Sciences, 2006, 100(5):398-410.

[7] Seoane L M, Al-massadi O, Lage M, Dieguez C, Casanueva F F. Ghrelin:from a GH-secretagogue to the regulation of food intake, sleep and anxiety. Pediatric Endocrinology Reviews, 2004, 1(suppl 3):432-437.

[8] Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa, K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature, 1999, 402(6762):656-660.

[9] 张爱玲, 张丽, 陈宏, 张良志, 蓝贤勇, 张春雷, 张存芳, 朱泽轶. 黄牛Ghrelin基因的克隆、表达及生物学活性检测. 生物工程学报, 2009, 25(1):23-28.

Zhang A L, Zhang L, Chen H, Zhang L Z, Lan X Y, Zhang C L, Zhang C F, Zhu Z Y. Cloning, prokaryotic expression of cattle Ghrelin gene and biological activity detection of the expressed protein. Chinese Journal of Biotechnology, 2009, 25(1):23-28. (in Chinese).

[10] Tschop M, Smiley D L, Heiman M L. Ghrelin induces adiposity in rodents. Nature, 2000, 407: 908-913.

[11] Asakawa A, Inui A, Kaga T, Yuzuriha H, Nagata T, Ueno N, Makino S, Fujimiya M, Niijima A, Fujino M A, Kasuga M. Ghrelin is an appetite-stimulatory signal from stomach with structural resemblance to motilin. Gastroentemlogy, 2001, 120(2):337-345.

[12] Li C C, Li K, Li J, Mo D L, Xu R F, Chen G H, Qiangba Y Z, Ji S L, Tang X H, Fan B, Zhu M J, Xiong T A, Guan X, Liu B. Polymorphism of ghrelin gene in twelve indigenous chicken breeds and its relationship with chicken growth traits. Asian-Australia Journal Animal Science, 2006, 19:153-159.

[13] 何丹林, 方梅霞, 聂庆华, 彭君瑜, 邓玉珍, 张细权. 鸡Ghrelin基因C2100T位点与生长和脂肪性状的相关性. 广东农业科学, 2004 (7):73-81.

He D L, Fang M X, Nie Q H, Peng J Y, Deng Y Z, Zhang X Q. Association of Ghrelin gene C2100T polymorphism with chicken growth and fat traits. Guangdong Agricultural Sciences, 2004 (7):73-81.(in Chinese).

[14] Fang M, Nie Q, Luo C, Zhang D, Zhang X. An 8 bp indel in exon 1 of Ghrelin gene associated with chicken growth. Domestic Animal Endocrinology, 2007, 32(3):216-225.

[15] Howard A D, Feighner S D, Cully D F, Arena J P, Liberator P A, Rosenblum C I, Hamelin M, Hreniuk D L, Palyha O C, Anderson J, Paress P S, Diaz C, Chou M, Liu K K, McKee K K, Pong S S, Chaung L Y, Elbrecht A, Dashkevicz M, Dean D C, Melillo D G, Patchett A A, Nargund R, Griffin P R, DeMartino J A, Gupta S K, Schaeffer J M, Smith R G, Heavens R, Rigby M. A receptor in pituitary and hypotha-lamus that functions in growth hormone release. Science, 1996, 273(5277):974-977.

[16] Shuto Y, Shibasaki T, Otagiri A, Kuriyama H, Ohata H, Tamura H, Kamegai J, Sugihara H, Oikawa S, Wakabayashi I. Hypothalamic growth hormone secretagogue receptor regulates growth hormone secretion, feeding, and adiposity. The Journal of Clinical Investigation, 2002, 109(11):1429-1436.

[17] Pantel J, Legendre M, Cabrol S, Hilal L, Hajaji Y, Morisset S, Nivot S, Pierre M, Luton V, Grouselle D, Kerdanet M, Kadiri A, Epelbaum J, Bouc Y L, Amselem S. Loss of constitutive activity of the growth hormone secretagogue receptor in familial short stature. The Journal of Clinical Investigation, 2006, 116(3):760-768.

[18] 宋桃伟, 罗卫星, 蔡惠芬, 刘彬, 孙岩岩. 山羊GHSR基因多态性及其与体重体尺性状的相关性研究.畜牧兽医学报, 2013, 44(11): 1717-1723.

Song T W, Luo W X, Cai H F, Liu B, Sun Y Y. Polymorphism of GHSR Gene and its relevance with body weight and body size traits in Goat. Acta Veterinaria et Zootechnica Sinica, 2013, 44(11):1717-1723. (in Chinese).

[19] Jin Q J, Fang X T, Yang L, Zhang C L, Sun J J, Chen D X, Shi X Y, Du Y, Lei C C, Chen H.Novel SNPs of the caprine growth hormone secretagogue receptor (GHSR) gene and their association with growth traits in goats. Biochemical Genetics, 2010, 48(9/10):847-856.

[20] 李敬瑞. 猪五个候选基因的遗传变异研究. 贵阳:贵州大学, 2011.

Li J R. Study on the genetic variation of five candidate genes in swine[D]. Guiyang: Guizhou University, 2011. (in Chinese).

[21] 李树珍, 万慧荣, 杨光. DNA池结合DHPLC和直接测序技术在江豚SNPs检测中的应用. 兽类学报, 2009, 29(2): 185-190.

Li S Z, Wan H R, Yang G. Application of DNA pooling in combination with DHPLC and direct sequencing in SNPs detection of the finless porpoise (Neophocaena phocaenoides). Acta Theriologica Sinica, 2009, 29(2):185-190. (in Chinese).

[22] 李稳, 李勇, 张龙超, 王立刚, 颜华, 王立贤. 猪多潜能性维持因子5基因遗传多态性及其与繁殖性状的关联分析. 畜牧兽医学报, 2011, 42(11):1513-1518.

Li W, Li Y, Zhang L C, Wang L G, Yan H, Wang L X. Genetic polymorphisms of the porcine developmental pluripotency associated 5 gene and its relationship with reproductive traits. Acta Veterinaria et Zootechnica Sinica, 2011, 42(11):1513-1518. (in Chinese).

[23] 淮永涛. 黄牛垂体关键转录因子SIX3和SIX6基因的遗传变异及其与生长性状关联的分析[D]. 杨凌: 西北农林科技大学, 2010.

Huai Y T. Genetic variation of pituitary gland transcriptional factors six3 &six6 genes in yellow cattle and their association with growth traits[D]. Yangling: Northwest Agriculture & Forestry University, 2010. (in Chinese).

[24] 方梅霞, 聂庆华, 何丹琳, 刘满清, 张细权. 四个鸡品种GHSR基因遗传多样性分析. 中国家禽科学研究进展, 2010, 312-316.

Fang M X, Nie Q H, He D L, Liu M Q, Zhang X Q. Analysis of genetic diversity of GHSR gene in four chicken breeds.Chinese Poultry Science Progress, 2010, 312-316. (in Chinese)

[25] 方梅霞, 李莹, 徐海平, 谢亮, 廖新悌, 梁敏, 聂庆华, 张细权. GHRL及其受体GHSR基因多态性与鸭生长及屠体性状的关联性. 畜牧兽医学报, 2011,42(1):18-24.

Fang M X, Li Y, Xu H P, Xie L, Liao X T, Liang M, Nie Q H, Zhang X Q. Associations of ghrelin(GHRL)and its receptor (GHSR) genes polymorphisms with duck growth and carcass traits. Acta Veterinaria et Zootechnica Sinica, 2011, 42(1):18-24. (in Chinese).

[26] 王欢, 高风英, 叶星, 卢迈新, 黄樟翰, 朱华平, 杨丽萍. 两种罗非鱼GHSR基因5'侧翼区域的克隆及序列分析//2009年中国水产学会学术年会论文集摘要. 中国水产学会, 2009, 11.

Wang H, Gao F Y, Ye X, Lu M X, Huang Z H, Zhu H P, Yang L P. Isolation, and characterization of 5’ flanking region of two tilapia// Summary of Proceedings of Annual Conference of China Fisheries Society in 2009. China Society of Fisheries, 2009, 11. (in Chinese)