儋州鸡FSHβ基因的单核苷酸多态性研究
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摘要
为了研究儋州鸡FSHβ基因单核苷酸多态性(SNP),试验采用PCR产物直接测序法,对60份儋州鸡DNA样本的FSHβ基因进行PCR扩展和双向测序,对测序结果组装后使用DNAMAN软件进行比对,人工核对后筛查SNP位点。结果表明:PCR扩增均能得到与预期相符的清晰条带;测序结果与GenBank中登录的原鸡FSHβ序列比对显示,为儋州鸡FSHβ基因的片段;SNP位点筛查显示,儋州鸡FSHβ基因中含有25个SNP位点,即C-130T、A-332C、T-439C、G-591A、C-956A、G-1 233A、G-1 386C、G-1 466A、T-1 542C、T-1 543A、C-1 703T、C-1 714T、T-1 834C、A-1 867G、A-1 960C、C-2 021T、G-2 285T、G-2 399T、T-2 528C、A-2 536G、T-2 607C、G-2 770A、A-3 229G、-665处插入AA及-2 566处缺失C,这25个SNP位点包含了5个内含子突变和20个外显子突变,其中G-591A和G-1 233A为编码区错义突变。
The aim of the present study was toinvestigate the single nucleotide polymorphisms(SNP) of the FSHβ gene in Danzhou chicken.PCR amplification and bidirectional sequencing of FSHβ gene from 60 Danzhou chicken DNA samples were performed by direct sequencing of PCR products. After the sequencing results were assembled, the DNAMAN software was used for comparison, and the SNP sites were screened by manual verification. The results showed that clear bands could be obtained by PCR amplification.The result of sequencing was compared with the sequence of FSHβ of chicken registered in GenBank, and the result showed that it was a fragment of FSHβ gene of Danzhou chicken.The screening of SNP loci showed that there were 25 SNP loci in the FSHβ gene of Danzhou chickens. They are C-130 T, A-332 C, T-439 C, G-591 A, C-956 A, G-1 233 A, G-1 386 C, G-1 466 A, T-1 542 C, T-1 543 A, C-1 703 T, C-1 714 T, T-1 834 C, A-1 867 G, A-1 960 C, C-2 021 T, G-2 285 T, G-2 399 T, T-2 528 C, A-2 536 G, T-2 607 C, G-2 770 A, A-3 229 G, insert AA at-665 site and deletion of C at-2 566 site.The 25 SNP loci contained 5 intron mutations and 20 exon mutations, of which G-591 A and G-1 233 A were missense mutations in the coding region.
The aim of the present study was toinvestigate the single nucleotide polymorphisms(SNP) of the FSHβ gene in Danzhou chicken.PCR amplification and bidirectional sequencing of FSHβ gene from 60 Danzhou chicken DNA samples were performed by direct sequencing of PCR products. After the sequencing results were assembled, the DNAMAN software was used for comparison, and the SNP sites were screened by manual verification. The results showed that clear bands could be obtained by PCR amplification.The result of sequencing was compared with the sequence of FSHβ of chicken registered in GenBank, and the result showed that it was a fragment of FSHβ gene of Danzhou chicken.The screening of SNP loci showed that there were 25 SNP loci in the FSHβ gene of Danzhou chickens. They are C-130 T, A-332 C, T-439 C, G-591 A, C-956 A, G-1 233 A, G-1 386 C, G-1 466 A, T-1 542 C, T-1 543 A, C-1 703 T, C-1 714 T, T-1 834 C, A-1 867 G, A-1 960 C, C-2 021 T, G-2 285 T, G-2 399 T, T-2 528 C, A-2 536 G, T-2 607 C, G-2 770 A, A-3 229 G, insert AA at-665 site and deletion of C at-2 566 site.The 25 SNP loci contained 5 intron mutations and 20 exon mutations, of which G-591 A and G-1 233 A were missense mutations in the coding region.
引文
[1] 李道劲.我国优质鸡育种的发展趋向[J].中国家禽, 2003,25(1):7-8.
[2] 黎寿丰, 王桂朝.我国优质鸡育种技术现状与创新方向探讨[J].中国家禽,2006, 28(15):5-9.
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[7] 王鹏, 管庆丰, 华蕊, 等. 促卵泡素和促黄体生成素对儋州鸡生殖激素调控规律的研究[J]. 中国畜牧兽医,2017,44(10):3029-3034.
[8] 石铁流.新技术能使DNA测序的成本降低多少[J].科学通报, 2017(19):2042-2046.
[9] CARR I M, VALLELEY E M, CORDERY S F, et al. Sequence analysis and editing for bisulphite genomic sequencing projects[J]. Nucleic Acids Res, 2007, 35(10):e79.
[10]HUNZICKER-DUNN M, MAIZELS E T. FSH signaling pathways in immature granulosa cells that regulate target gene expression. Branching out from protein kinase A[J]. Cell Signal, 2006,18(9): 1351-1359.
[11]MCELROY A P, CALDWELL D J, PROUDMAN J A, et al. Modulation of in vitro DNA synthesis in the chicken ovarian granulosa cell follicular hierarchyby follicle-stimulating hormone and luteinizing hormone[J]. Poult Sci, 2004, 83(3): 500-506.
[12]JIN Y, ZHANG C, LIN X, et al. Prostaglandin involvement in follicle-stimulating hormone-induced proliferation of granulosa cells from chicken prehierarchical follicles[J]. Prostaglandins Other Lipid Mediat, 2006, 81(1/2): 45-54.
[13]ORISAKA M, MIZUTANI T, TAJIMA K, et al. Effects of ovarian theca cells on granulosa cell differentiation during gonadotropin-independent follicular growth in cattle[J]. Mol Reprod Dev, 2006, 73(6):737-744.
[14]PALERMO R. Differential actions of FSH and LH during folliculogenesis[J]. Reprod Biomed Online, 2007,15(3):326-337.
[15]郑心力, 刘海隆, 黄丽丽, 等. 五指山猪FSHβ基因克隆测序及生物信息学分析[J]. 黑龙江畜牧兽医, 2016(09上):93-95.
[16]宋敏艳, 高文春, 罗在仁, 等.德宏水牛及杂交后代FSHβ基因多态性与繁殖性能的关系[J].中国牛业科学, 2013, 39(5): 26-32.
[17]梁琛,储明星,张建海,等. FSHβ基因PCR-SSCP多态性及其与济宁青山羊高繁殖力关系的研究[J]. 遗传, 2006, 28(9): 1071-1077.
[18]叶卉, 刘云龙, 邹秉杰, 等.焦磷酸测序中PCR引物与测序引物的设计[J].分析化学, 2013, 41(5):744-748.
[19]宋艳斌, 马文丽, 郑文岭. DNA测序中常见影响因素的研究[J]. 生物技术通报, 2005(1):44-46.
[20]王青娟, 李同明, 尹逊河, 等.文昌鸡促卵泡激素β基因内含子1的SNP分析及其与早期产蛋性能的相关性研究[J]. 中国畜牧兽医, 2012, 39(12):117-121.
[2] 黎寿丰, 王桂朝.我国优质鸡育种技术现状与创新方向探讨[J].中国家禽,2006, 28(15):5-9.
[3] SHEN S T,YU Y L.Cloning and gene expression of a cDNA for the chicken follicle-stimulating hormone (FSH)-beta-subunit[J]. Gen Comp Endocrinol, 2002, 125(3):375-386.
[4] LEWIS P D, CICCONE N, SHARP P J, et al. Light intensity can influence plasma FSH and age at sexual maturity in domestic pullets[J].Br Poult Sci, 2005, 46(4):506-509.
[5] CICCONE N A, SHARP P J, WILSON P W, et al. Changes in reproductive neuroendocrine mRNAs with decreasing ovarian function in ageing hens[J]. Gen Comp Endocrinol, 2005, 144(1):20-27.
[6] 王鹏,管庆丰,文福,等.儋州鸡不同产蛋时期生殖激素分泌规律的研究[J].中国家禽,2017, 39(4):8-11.
[7] 王鹏, 管庆丰, 华蕊, 等. 促卵泡素和促黄体生成素对儋州鸡生殖激素调控规律的研究[J]. 中国畜牧兽医,2017,44(10):3029-3034.
[8] 石铁流.新技术能使DNA测序的成本降低多少[J].科学通报, 2017(19):2042-2046.
[9] CARR I M, VALLELEY E M, CORDERY S F, et al. Sequence analysis and editing for bisulphite genomic sequencing projects[J]. Nucleic Acids Res, 2007, 35(10):e79.
[10]HUNZICKER-DUNN M, MAIZELS E T. FSH signaling pathways in immature granulosa cells that regulate target gene expression. Branching out from protein kinase A[J]. Cell Signal, 2006,18(9): 1351-1359.
[11]MCELROY A P, CALDWELL D J, PROUDMAN J A, et al. Modulation of in vitro DNA synthesis in the chicken ovarian granulosa cell follicular hierarchyby follicle-stimulating hormone and luteinizing hormone[J]. Poult Sci, 2004, 83(3): 500-506.
[12]JIN Y, ZHANG C, LIN X, et al. Prostaglandin involvement in follicle-stimulating hormone-induced proliferation of granulosa cells from chicken prehierarchical follicles[J]. Prostaglandins Other Lipid Mediat, 2006, 81(1/2): 45-54.
[13]ORISAKA M, MIZUTANI T, TAJIMA K, et al. Effects of ovarian theca cells on granulosa cell differentiation during gonadotropin-independent follicular growth in cattle[J]. Mol Reprod Dev, 2006, 73(6):737-744.
[14]PALERMO R. Differential actions of FSH and LH during folliculogenesis[J]. Reprod Biomed Online, 2007,15(3):326-337.
[15]郑心力, 刘海隆, 黄丽丽, 等. 五指山猪FSHβ基因克隆测序及生物信息学分析[J]. 黑龙江畜牧兽医, 2016(09上):93-95.
[16]宋敏艳, 高文春, 罗在仁, 等.德宏水牛及杂交后代FSHβ基因多态性与繁殖性能的关系[J].中国牛业科学, 2013, 39(5): 26-32.
[17]梁琛,储明星,张建海,等. FSHβ基因PCR-SSCP多态性及其与济宁青山羊高繁殖力关系的研究[J]. 遗传, 2006, 28(9): 1071-1077.
[18]叶卉, 刘云龙, 邹秉杰, 等.焦磷酸测序中PCR引物与测序引物的设计[J].分析化学, 2013, 41(5):744-748.
[19]宋艳斌, 马文丽, 郑文岭. DNA测序中常见影响因素的研究[J]. 生物技术通报, 2005(1):44-46.
[20]王青娟, 李同明, 尹逊河, 等.文昌鸡促卵泡激素β基因内含子1的SNP分析及其与早期产蛋性能的相关性研究[J]. 中国畜牧兽医, 2012, 39(12):117-121.