山羊LF基因序列分析及SNPs研究
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摘要
本试验根据GenBank上已发表的牛的乳铁蛋白(Lactoferrin, LF)基因DNA序列和山羊mRNA序列进行引物设计,扩增山羊LF基因的部分5’UTR至外显子17序列片断,测序总长度为30306bp,已提交到GenBank,序列号为FJ609300。为比较反刍动物LF基因遗传变异与分化,同时测定了绵羊LF基因相应序列30340bp,序列号为:FJ541507。
根据测序结果检测到山羊LF基因序列上共30个突变位点,其中处于外显子的有1个,内含子的29个。依据序列号FJ609300上的位置来命名这些突变位点,处于外显子上的1个点突变为7605C>T,引起氨基酸Arg136Trp突变。处于内含子上的29个突变位点分别为2428C>G、2761C>G、6053A>G、6538A>C、7363A>G、8642C>T、8655C>T、9284C>T、13719C>T、15783A>G、15820A>G、16820A>G、17774A>T、17775A>G、17793A>G、17808A>G、17855C>G、18266C>G、18428A>G、18930A>G、19764C>T、19997C>T、20740A>G、20989C>T、21146C>T、21281C>G、23146C>T、24300C>T、24720A>G、25249A>G。同时检测到绵羊LF基因序列上16个突变位点,都是内含子变异,依据序列号FJ541507上的位置来命名这些突变位点分别为:2432C>T、2764C>G、4197C>T、4274A>T、4633C>T、11038C>T、13045A>G、13075A>G、13482A>G、18072A>G、20660C>T、21022C>T、21625T>G、21706T>G、22583T>G、22594C>T。在山羊DNA序列中发现21个短散布元件和14个长散布元件。在绵羊DNA序列中发现19个短散布元件和14个长散布元件。另外,在山羊和绵羊DNA序列中各发现5个简单重复序列(SSR)。
7605C>T位点上检测到C和T两个等位基因。所研究的10个中国地方山羊品种/品系(承德无角山羊、成都麻羊、济宁青山羊、雷州山羊、辽宁绒山羊、南江黄羊黑色系、南江黄羊高繁系、唐山奶山羊、内蒙古绒山羊、武安山羊)共300个个体。T等位基因频率(50.12%)稍高于C等位基因(49.88%)。从基因型分布看,10个中国地方山羊群体的基因型频率在各群体间差异较大。遗传多样性分析表明,10个中国地方山羊在7605C>T位点具有较丰富的遗传多样性。群体遗传分化分析表明,10个中国地方山羊群体间的平均基因流为2.9433,平均遗传分化指数为0.0783。
本试验获得了山羊和绵羊LF基因完整编码区(CDS)序列,另外,从GenBank获得了60条不同物种LF基因完整CDS序列,比对分析LF基因在物种间和物种内的遗传变异和分化。大部分物种终止密码子使用TAA,(1个人变异个体使用TAG),而小鼠使用TGA( 1个变异体使用TAA)。物种间长度变异较大(2055bp~2190bp)。根据美国国家生物技术信息中心(NCBI)分类法,相近物种的LF基因的CDS序列长度相同或相差较小。挪威鼠最长为2190bp,是由于终止密码子突变(TAA>GAA)导致的66bp的延长。一个黑猩猩变异体(XM_001150238)LF基因CDS长度最短为2055bp,是由于81bp缺失片段造成的。物种内也存在长度变异,狗长度变异最复杂,其它物种LF基因CDS的变异较小。遗传多样性分析发现多数物种LF基因具有较丰富的遗传多样性,群体间的遗传多样性大于群体内。通过物种内的LF氨基酸序列比对,发现了新的氨基酸变异位点(人8个、小鼠6个、山羊2个、牛10个、猪20个、绵羊3个)。物种间的遗传分化明显,系统发生树所显示的物种的聚类关系与NCBI物种的分类学一致。
On the basis of cattle DNA and goat mRNA lactoferrin (LF) gene sequences from GenBank, partial 5’UTR to exon 17 of goat LF gene fragments was amplified. The total length of determined sequence was 30306bp, which had been handed out to GenBank, the accession number was FJ609300. In order to compareing the genetic diversity and differentiation among ruminant, sheep LF gene was sequenced (30340bp), and the accession number was FJ541507.
30 point mutations were found in goat LF gene sequence, of which 1 was in exon, and 29 was in intron. The mutation in exon was 7605C>T resulting in Arg136Trp. The 29 mutation in intron were 2428C>G, 2761C>G, 6053A>G, 6538A>C, 7363A>G, 8642C>T, 8655C>T, 9284C>T, 13719C>T, 15783A>G, 15820A>G, 16820A>G, 17774A>T, 17775A>G, 17793A>G, 17808A>G, 17855C>G, 8266C>G, 18428A>G, 18930A>G, 19764C>T, 19997C>T, 20740A>G, 20989C>T, 21146C>T, 21281C>G, 23146C>T, 24300C>T, 24720A>G and 25249A>G. Meanwhile, 16 point mutations were detected in LF gene sequence of sheep, which all were in intron. The 16 mutation in intron were 2432C>T, 2764C>G, 4197C>T, 4274A>T, 4633C>T, 11038C>T, 13045A>G, 13075A>G, 13482A>G, 18072A>G, 20660C>T, 21022C>T, 21625T>G, 21706T>G, 22583T>G and 22594C>T. 21 short interspersed nuclear elements and 14 long interspersed nuclear elements were detected in LF gene of goat, and 19 short interspersed nuclear elements and 14 long interspersed nuclear elements were detected in LF gene of sheep. Moreover, 5 simple sequence repeats (SSR) was discovered in LF gene of goat and sheep, respectively.
We determined a missense mutation C→T at position 7605. Meanwhile the PCR-RFLP of this mutation was performed in 300 individuals from 10 Chinese indigenous goat breeds/strain(sChengde polled goat, Chengdu Ma goat, Jining gray goat, Leizhou goat, Liaoning cashmere goat, two strains of Nanjiang brown goat, Tangshan dairy goat, Neimenggu cashmere goat and Wuan goat). The results showed that the gene frequency of allele T (50.12%) was little higher and that of allele C (49.88%) and the difference of genotypes was great among goat populations. The 7605C>T site performed higher genetic diversity in goat populations. The genetic differentiation was 0.0783 and gene flow was 2.9433 among 10 goat populations.
The coding regions (CDS) sequence of LF gene of goat was obtained by splicing in this study, and 60 sequences from 11 species were studied to investigate its evolution and differentiation within and among species. Most species use TAA as stop codon for the LF gene, with only one human variation (DQ892855) using TAG. Mus musculus uses TGA, except for one variation (CT010339) with TAG. It was shown that the length of the LF gene with the complete CDS varies greatly among species, ranging from 2055 to 2190bp. Species with close relationships according to the taxonomy in the National Center for Biotechnology Information (NCBI) have a similar length of the LF gene. The longest length (2190bp) of the LF gene, in Rattus norvegicus, is due to the stop codon mutation of TAA to GAA resulting in the 66 bp elongation. And the shortest length (2055bp) is one variant type of Pan roglodytes (XM_001150238) due to an 81bp deletion. There was length variation within species. Intricate length variations were in Canis familiaris with three variant regions, and simple length variations was in other species. Most species had abundant genetic diversity of LF gene, and genetic diversity was higher among species than within species. Novel amino acid variation sites were detected within several species (8 in Homo sapiens, 6 in Mus musculus, 2 in Capra hircus, 3 in Ovis aries, 10 in Bos Taurus and 20 in Sus scrofa). Differentiation of the LF gene was obvious among species, and the clustering result was consistent with the taxonomy in NCBI.
根据测序结果检测到山羊LF基因序列上共30个突变位点,其中处于外显子的有1个,内含子的29个。依据序列号FJ609300上的位置来命名这些突变位点,处于外显子上的1个点突变为7605C>T,引起氨基酸Arg136Trp突变。处于内含子上的29个突变位点分别为2428C>G、2761C>G、6053A>G、6538A>C、7363A>G、8642C>T、8655C>T、9284C>T、13719C>T、15783A>G、15820A>G、16820A>G、17774A>T、17775A>G、17793A>G、17808A>G、17855C>G、18266C>G、18428A>G、18930A>G、19764C>T、19997C>T、20740A>G、20989C>T、21146C>T、21281C>G、23146C>T、24300C>T、24720A>G、25249A>G。同时检测到绵羊LF基因序列上16个突变位点,都是内含子变异,依据序列号FJ541507上的位置来命名这些突变位点分别为:2432C>T、2764C>G、4197C>T、4274A>T、4633C>T、11038C>T、13045A>G、13075A>G、13482A>G、18072A>G、20660C>T、21022C>T、21625T>G、21706T>G、22583T>G、22594C>T。在山羊DNA序列中发现21个短散布元件和14个长散布元件。在绵羊DNA序列中发现19个短散布元件和14个长散布元件。另外,在山羊和绵羊DNA序列中各发现5个简单重复序列(SSR)。
7605C>T位点上检测到C和T两个等位基因。所研究的10个中国地方山羊品种/品系(承德无角山羊、成都麻羊、济宁青山羊、雷州山羊、辽宁绒山羊、南江黄羊黑色系、南江黄羊高繁系、唐山奶山羊、内蒙古绒山羊、武安山羊)共300个个体。T等位基因频率(50.12%)稍高于C等位基因(49.88%)。从基因型分布看,10个中国地方山羊群体的基因型频率在各群体间差异较大。遗传多样性分析表明,10个中国地方山羊在7605C>T位点具有较丰富的遗传多样性。群体遗传分化分析表明,10个中国地方山羊群体间的平均基因流为2.9433,平均遗传分化指数为0.0783。
本试验获得了山羊和绵羊LF基因完整编码区(CDS)序列,另外,从GenBank获得了60条不同物种LF基因完整CDS序列,比对分析LF基因在物种间和物种内的遗传变异和分化。大部分物种终止密码子使用TAA,(1个人变异个体使用TAG),而小鼠使用TGA( 1个变异体使用TAA)。物种间长度变异较大(2055bp~2190bp)。根据美国国家生物技术信息中心(NCBI)分类法,相近物种的LF基因的CDS序列长度相同或相差较小。挪威鼠最长为2190bp,是由于终止密码子突变(TAA>GAA)导致的66bp的延长。一个黑猩猩变异体(XM_001150238)LF基因CDS长度最短为2055bp,是由于81bp缺失片段造成的。物种内也存在长度变异,狗长度变异最复杂,其它物种LF基因CDS的变异较小。遗传多样性分析发现多数物种LF基因具有较丰富的遗传多样性,群体间的遗传多样性大于群体内。通过物种内的LF氨基酸序列比对,发现了新的氨基酸变异位点(人8个、小鼠6个、山羊2个、牛10个、猪20个、绵羊3个)。物种间的遗传分化明显,系统发生树所显示的物种的聚类关系与NCBI物种的分类学一致。
On the basis of cattle DNA and goat mRNA lactoferrin (LF) gene sequences from GenBank, partial 5’UTR to exon 17 of goat LF gene fragments was amplified. The total length of determined sequence was 30306bp, which had been handed out to GenBank, the accession number was FJ609300. In order to compareing the genetic diversity and differentiation among ruminant, sheep LF gene was sequenced (30340bp), and the accession number was FJ541507.
30 point mutations were found in goat LF gene sequence, of which 1 was in exon, and 29 was in intron. The mutation in exon was 7605C>T resulting in Arg136Trp. The 29 mutation in intron were 2428C>G, 2761C>G, 6053A>G, 6538A>C, 7363A>G, 8642C>T, 8655C>T, 9284C>T, 13719C>T, 15783A>G, 15820A>G, 16820A>G, 17774A>T, 17775A>G, 17793A>G, 17808A>G, 17855C>G, 8266C>G, 18428A>G, 18930A>G, 19764C>T, 19997C>T, 20740A>G, 20989C>T, 21146C>T, 21281C>G, 23146C>T, 24300C>T, 24720A>G and 25249A>G. Meanwhile, 16 point mutations were detected in LF gene sequence of sheep, which all were in intron. The 16 mutation in intron were 2432C>T, 2764C>G, 4197C>T, 4274A>T, 4633C>T, 11038C>T, 13045A>G, 13075A>G, 13482A>G, 18072A>G, 20660C>T, 21022C>T, 21625T>G, 21706T>G, 22583T>G and 22594C>T. 21 short interspersed nuclear elements and 14 long interspersed nuclear elements were detected in LF gene of goat, and 19 short interspersed nuclear elements and 14 long interspersed nuclear elements were detected in LF gene of sheep. Moreover, 5 simple sequence repeats (SSR) was discovered in LF gene of goat and sheep, respectively.
We determined a missense mutation C→T at position 7605. Meanwhile the PCR-RFLP of this mutation was performed in 300 individuals from 10 Chinese indigenous goat breeds/strain(sChengde polled goat, Chengdu Ma goat, Jining gray goat, Leizhou goat, Liaoning cashmere goat, two strains of Nanjiang brown goat, Tangshan dairy goat, Neimenggu cashmere goat and Wuan goat). The results showed that the gene frequency of allele T (50.12%) was little higher and that of allele C (49.88%) and the difference of genotypes was great among goat populations. The 7605C>T site performed higher genetic diversity in goat populations. The genetic differentiation was 0.0783 and gene flow was 2.9433 among 10 goat populations.
The coding regions (CDS) sequence of LF gene of goat was obtained by splicing in this study, and 60 sequences from 11 species were studied to investigate its evolution and differentiation within and among species. Most species use TAA as stop codon for the LF gene, with only one human variation (DQ892855) using TAG. Mus musculus uses TGA, except for one variation (CT010339) with TAG. It was shown that the length of the LF gene with the complete CDS varies greatly among species, ranging from 2055 to 2190bp. Species with close relationships according to the taxonomy in the National Center for Biotechnology Information (NCBI) have a similar length of the LF gene. The longest length (2190bp) of the LF gene, in Rattus norvegicus, is due to the stop codon mutation of TAA to GAA resulting in the 66 bp elongation. And the shortest length (2055bp) is one variant type of Pan roglodytes (XM_001150238) due to an 81bp deletion. There was length variation within species. Intricate length variations were in Canis familiaris with three variant regions, and simple length variations was in other species. Most species had abundant genetic diversity of LF gene, and genetic diversity was higher among species than within species. Novel amino acid variation sites were detected within several species (8 in Homo sapiens, 6 in Mus musculus, 2 in Capra hircus, 3 in Ovis aries, 10 in Bos Taurus and 20 in Sus scrofa). Differentiation of the LF gene was obvious among species, and the clustering result was consistent with the taxonomy in NCBI.
引文
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