鹅胰岛素样生长因子-Ⅰ（IGF-Ⅰ）基因克隆测序及多态性研究
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摘要
本实验以扬州鹅为素材,对鹅胰岛素样生长因子-Ⅰ(IGF-Ⅰ)基因组部分序列进行了克隆、测序,并用PCR-SSCP的方法对其多态性进行了检测。主要结果如下:
1.得到了鹅IGF-Ⅰ基因的5’调控区、外显子1、4及内含子1、3的全部或部分序列,所测序得到的内含子1、内含子3和外显子4序列已上传至Genbank,序列号分别为EU072031、EU111743。本研究首次得到了鹅IGF-Ⅰ基因的内含子1和3序列。序列比对发现,鹅IGF-Ⅰ基因与鸡、鸭同源性较高,无论是外显子还是内含子都大于80%(除内含子3),外显子1与鸡、鹌鹑、火鸡、鸭、鸵鸟、鹅等禽类同源性在98%以上,与梅花鹿、山羊、猪、牛、北极狐、绵羊、小鼠和人等哺乳动物同源性在89%～94%之间,与乌颊鱼、罗非鱼的同源性在60%～65%之间。对鹅5’调控区和外显子1序列进行转录因子结合位点和启动子区域预测,发现有多个转录因子结合位点和一个启动子区域。进化分析表明IGF-Ⅰ基因的进化与物种进化相一致,反映了IGF-Ⅰ基因在进化中的保守性及生理功能的重要性。
2.根据鹅IGF-Ⅰ基因的5’调控区、外显子1和内含子1部分序列设计了7对引物,用PCR-SSCP的方法对其进行单核苷酸多态性(SNP)检测。结果未发现多态性。
In this study, the partial genomic sequences of Insulin-like Growth Factor-ⅠGene (IGF-Ⅰ) of yangzhou goose were cloned and sequenced, and their DNA polymorphism was detected by PCR-SSCP. Main results are as follows:
1. Complete sequences of exon 1, intron 1 and partial sequences of 5’-UTR, intron 3 and exon 4 were obtained in the study. The sequences of intron 1, intron 3 and exon 4 were submitted to Genbank, with accession NO. EU111743 and EU072031. The sequences of intron 1 and 3 of goose IGF-Ⅰgene were reported for the first time. By analysis of homology, the homology of chicken, duck and goose is more than 80% (except intron 3). And the homology of chicken, quail, turkey, duck, ostrich and goose IGF-Ⅰexon 1 is above 98%. The homology between exon 1 and sika deer, goat, pig, cattle, arctic fox, sheep, mouse and human is between 89% and 94%. And the homology between exon 1 and gilthead, tilapia is between 60% and 65%. Several transcription factor binding sites and one promoter was found in 5’–UTR and exon 1 after prediction. The evolution analysis indicated the high identities both in species and IGF-Ⅰgene. The gene was conservation in the process of evolution and had important physiological function.
2. Single nucleotide polymorphism (SNP) of IGF-Ⅰgene were detected with seven pairs of primers by PCR-SSCP in 5’-UTR, exon 1 and intron 1 of IGF-Ⅰgene in yangzhou goose. No single nucleotide polymorphism was found.
1.得到了鹅IGF-Ⅰ基因的5’调控区、外显子1、4及内含子1、3的全部或部分序列,所测序得到的内含子1、内含子3和外显子4序列已上传至Genbank,序列号分别为EU072031、EU111743。本研究首次得到了鹅IGF-Ⅰ基因的内含子1和3序列。序列比对发现,鹅IGF-Ⅰ基因与鸡、鸭同源性较高,无论是外显子还是内含子都大于80%(除内含子3),外显子1与鸡、鹌鹑、火鸡、鸭、鸵鸟、鹅等禽类同源性在98%以上,与梅花鹿、山羊、猪、牛、北极狐、绵羊、小鼠和人等哺乳动物同源性在89%～94%之间,与乌颊鱼、罗非鱼的同源性在60%～65%之间。对鹅5’调控区和外显子1序列进行转录因子结合位点和启动子区域预测,发现有多个转录因子结合位点和一个启动子区域。进化分析表明IGF-Ⅰ基因的进化与物种进化相一致,反映了IGF-Ⅰ基因在进化中的保守性及生理功能的重要性。
2.根据鹅IGF-Ⅰ基因的5’调控区、外显子1和内含子1部分序列设计了7对引物,用PCR-SSCP的方法对其进行单核苷酸多态性(SNP)检测。结果未发现多态性。
In this study, the partial genomic sequences of Insulin-like Growth Factor-ⅠGene (IGF-Ⅰ) of yangzhou goose were cloned and sequenced, and their DNA polymorphism was detected by PCR-SSCP. Main results are as follows:
1. Complete sequences of exon 1, intron 1 and partial sequences of 5’-UTR, intron 3 and exon 4 were obtained in the study. The sequences of intron 1, intron 3 and exon 4 were submitted to Genbank, with accession NO. EU111743 and EU072031. The sequences of intron 1 and 3 of goose IGF-Ⅰgene were reported for the first time. By analysis of homology, the homology of chicken, duck and goose is more than 80% (except intron 3). And the homology of chicken, quail, turkey, duck, ostrich and goose IGF-Ⅰexon 1 is above 98%. The homology between exon 1 and sika deer, goat, pig, cattle, arctic fox, sheep, mouse and human is between 89% and 94%. And the homology between exon 1 and gilthead, tilapia is between 60% and 65%. Several transcription factor binding sites and one promoter was found in 5’–UTR and exon 1 after prediction. The evolution analysis indicated the high identities both in species and IGF-Ⅰgene. The gene was conservation in the process of evolution and had important physiological function.
2. Single nucleotide polymorphism (SNP) of IGF-Ⅰgene were detected with seven pairs of primers by PCR-SSCP in 5’-UTR, exon 1 and intron 1 of IGF-Ⅰgene in yangzhou goose. No single nucleotide polymorphism was found.
引文
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[5]欧阳建华,孙汉,林树茂等.鸡胰岛素样生长因子-1基因的遗传多态性与其体重的关系[J].畜牧兽医学报, 2003, 34(6): 525～529.
[6]范刚.新扬州鸡IGF-1基因多态性与早期生长速度的关系研究[J].中国家禽, 2004, 26(24): 10～13.
[7] Zhou. H, Jimenez. N, Villalba. D, et al. Identification of three SNP in the chicken IGF-1 and IGF-2 gene and their associations with growth, body composition, skeletonitegrity, and metabolic traits in chickens [J]. Poultry Sci., 2005, 84: 212～219.
[8] M. H. Kim, D. S. Seo, Y. Ko. Relationship between egg productivity and insulin-like growth factor-1 genotypes in Korean Native Ogol Chickens [J]. Poultry Sci., 2004, 83: 1203～1208.
[9]万秋蓓.鸡类胰岛素样生长因子-Ⅰ(IGF-Ⅰ)基因多态性与蛋用性状关系及其表达的研究: [硕士学位论文][D].江苏:扬州大学, 2007.
[10]雷明明,张德祥,杨关福等. IGFs和IGFBP-2基因的单核苷酸多态及其与鸡生长、屠宰性能的相关分析.中国畜牧兽医学会2004年学术年会论文集[C] , 259～263.
[11]周锦玉.吉林白鹅IGF-Ⅰ基因的SNP与产绒性状的相关性研究: [硕士学位论文][D].吉林:吉林农业大学, 2007.
[12]王强.鸡IGF-Ⅰ基因PCR-SSCP分析及其与肉用性状关系的研究: [硕士学位论文][D].江苏:扬州大学, 2006.
[13]王志跃,范刚,杨海明等.新扬州鸡IGF-1基因多态性与早期生长速度关系的研究[J].中国家禽, 2004, 26(24): 9～12.
[2] Orita M, Iwahana H, Kanazawa H, et al. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms [J]. Proc Natl Acad Sci USA, 1989, 86: 2766~2770.
[3] Orita M, Suzuki Y, Sekiya T, et al. A rapid and sensitive detection of point mutations and genetic polymorphism using polymerase chain reaction [J]. Genomics, 1989, 5: 874~879.
[4] Hoshino S, Kimura A, Fukuda Y, et al. Polymerase chain reaction-single-strand conformation polymorphism analysis of polymorphism in DPA1 and DPB1 genes: a simple, economical, and rapid method for histocompatibility testing [J]. Hum Immunol, 1992, 33(2): 98~107.
[5] Salmon W D, Daughaday W H. A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro [J]. J Lab Clin Med, 1957, 49: 825~836.
[6] Pierson R W Jr, Temin H M, et al. The partial purification from calf serum of a fraction with multiplication-stimulating activity for chicken fibroblasts in cell culture and with non-suppressible insulin-like activity [J]. J Cell Physiol, 1972, 79 (3): 319~330.
[7] Mohan S, Baylink D J, Pettis J L, et al. Editoral: Insulin-like growth factor (IGF) binding protein sinserum Do they have additional roles be sides modulating the endocrine IGF actions [J]. J Clin Endocrinol Metab, 1996, 81(9): 3817~3820.
[8] Gluckman P D, Douglas R G, Ambler GR, et al. The endocrine role of insulin-like growth factor 1 [J]. Acta Paediatr Scand, 1991, 372 [Sup pl]: 97~105.
[9] Daughaday W H, Phillips L S, Mueller M C, et al. The effects of insulin and growth hormone on the release of soma to medin by the isolated rat liver [J]. Endocrinology, 1976, 98(4): 1214~1219.
[10]路聪哲. IGF-1与IGFBP1在Graves病糖代谢中的作用[J].国外医学内分泌学分册, 2001, 21(4): 212~213.
[11] Ernst M, Froesch E R. Growth hormone dependent stimulation of osteoblast like cells in serum-free cultures via local synthesis of insulin-like growth factor 1 [J]. Biochem Biophys Res Commun, 1988, 151 (1): 142~147.
[12] Thissen J P, Pucilowska J B, Underwood L E. Differential regulation of insulin-like growth factor 1 (IGF-1) and IGF binding protein-1 messenger ribonucleic acids by amino acid availability and growth hormone in rat hepatocyte primary culture [J]. Endocrinology, 1994, 134(6): 1570~1576.
[13] Maiter D, Mase M, Underwood L D, et al. Early changes in serum concentrations of somato medin-C induced by dietary protein deprivation in rats: contributions of growth hormone receptor and post receptor defects [J]. J Endocrinol. 1988, 118(1): 113~120.
[14] Malmof K, Arrhenius-Nyberg V, Saxerholt H, et al. The role of insulin, insulin-like growth factor 1 and growth hormone in counteracting dexamethasone induceed nitrogen wasting in rats [J]. Horm Metab Res, 1997, 29: 20~24.
[15] Salomom F, Cuneo R C, Hesp R, et al. The effects of treatment with recombineant human growth hormone on body composition and metabolism in adults with growth hormone deficiency [J]. N Engl J Med, 1989, 321(22): 1797~1803.
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