猪BMP7基因启动子多态性及其与繁殖性状关联性分析
摘要
骨形态发生蛋白7(BMP7)是BMP家族成员之一,具有多方面的生物学活性,对动物繁殖机能具有重要的调控作用。因目前尚未见猪BMP7基因遗传多态性分析的文献报道,所以本研究首次对猪BMP7基因启动子部位的遗传变异特性及其与猪繁殖性状相关性进行了分析。首先提取猪毛囊基因组DNA,选取不同品种极端性状的个体100头,构建极端性状混合DNA池,通过测序的方法,对猪BMP7基因启动子的遗传变异位点进行筛选。在此基础上,应用PCR-RFLP技术,对3个品种255头猪BMP7基因启动子区的4个SNP位点T-3722C、A-3684G、C-3522G、A-1027G进行群体遗传学分析,并用SPSS16.0分析这4个SNP位点对猪繁殖性状的影响。主要研究结果如下:
(1)应用猪毛囊提取的基因组DNA池,通过测序,首次发现启动子区存在39个多态位点。
(2)以猪BMP7基因测序结果为基础,应用PCR-RFLP技术首次检测启动子区4个SNP位点T-3722C、A-3684G、C-3522G和A-1027G。测序结果表明4个SNP位点分别为T/C突变、A/G突变、C/G突变和A/G突变,4个位点都有2个等位基因,3种基因型。遗传变异分析发现,T-3722C位点T是优势等位基因(0.67),优势基因型为TC型(0.48),该位点处于Hardy-Weinberg平衡状态,多态信息含量(PIC)、杂合度(He)、有效等位基因数(Ne)分别为0.34、0.44、1.80,属中度多态。A-3684G位点G是优势等位基因(0.84),优势基因型为GG型(0.76),该位点处于Hardy-Weinberg非平衡状态,PIC、He、Ne分别为0.23、0.27、1.36,属低度多态。C-3522G位点C是优势等位基因(0.71),优势基因型为CG型(0.48),该位点处于Hardy-Weinberg非平衡状态,PIC、He、Ne分别为0.33、0.41、1.70,属中度多态。A-1027G位点A是优势等位基因(0.81),优势基因型为AA型(0.67),该位点处于Hardy-Weinberg平衡状态,PIC、He、Ne分别为0.26、0.31、1.44,属中度多态。
(3)将检测到的4个SNP位点与繁殖性状相关的生产数据进行关联分析。结果显示,A-3684G位点与猪繁殖性状进行关联分析均表现无显著关联(P>0.05)。T-3722C位点与产活仔数、出生窝重及21日龄窝重显著相关(P<0.05);TT和TC型产产活仔数、出生窝重及21日龄窝重均极显著高于CC型。C-3522G位点与21日龄窝重极显著相关(P<0.01),CC和CG型极显著低于GG型。A-1027G位点与产活仔数及出生窝重显著相关(P<0.05),AA和AG型显著高于GG型。应用TFsearch等在线程序对猪BMP7启动子进行分析,预测到3个与繁殖性状显著关联的SNP位点均正好位于或靠近某些重要转录因子结合位点,而与繁殖性状无关联的位点则不在可能的转录因子结合位点区域。
基于以上结果,可得出如下结论:
(1)对猪BMP7基因启动子区域多态位点进行了筛选,发现了39个位点。
(2)猪BMP7基因启动子区域遗传变异性丰富,可能对基因表达乃至表型产生较大的影响。
(3)4个位点中T-3722C、C-3522G和A-1027G 3个位点与猪若干繁殖性状显著关联,它们可能通过改变启动子的转录活性而影响BMP7基因表达及相应的表型。这些多态位点可望作为分子标记用于育种中的辅助选择。
Bone morphogenetic protein 7 is a member of the family of bone morphogenetic proteins. The multifunctional protein also plays an important role in formation of bone and cartilage, and is also involved in the regulation of mammalian reproduction. No reports on the genetic diversity analysis have been found so far in the studies of porcine BMP7 gene, thus the present study took this gene as a target gene for the pig reproductive traits. First, a DNA pool was build using DNA samples from 100 sows of different breeds, and sequenced to screen the putative SNPs occurred in the promoter region. Then after, the PCR-RFLP was employed to genotype the total 255 sows and gilts including 26 Landrace, 147 Largewhite, 82 Doroc at the 4 loci (T-3722C, A-3684G, C-3522G, A-1027G). The main results are as follows:
(1) A total 39 polymorphic loci, including of 3 deletes and 36 signal nucleotide polymorphisms, were pick out for the first time in the studies of this gene.
(2) The genotyping for the 4 variations found reveled that each of these loci occurred in the population studied with the full 3 genotypes. Of 4 sites, T-3722C was dominated by the allele T and genotype TC, with an allele frequency and genotype frequency of 0.67 and 0.48, respectively. The further examination showed that this locus coincided with the Hardy-Weinberg equilibrium, with a polymorphic information content(PIC) ,heterozygosity (He)and effective allele number(Ne) of 0.34,0.44 and 1.80 in the population of interest, respectively. At the locus A-3684G, the dominated allele and genotype were G (0.84) and GG (0.76), respectively. Whereas, this locus was not in the balance of Hardy-Weinberg, and PIC, He and Ne were 0.23, 0.27 and 1.36, respectively. For another locus C-3522G, the dominances were C and CG, with the frequencies of 0.71 and 0.48, respectively. The locus C-3522G was also not consistent to the Hardy-Weinberg equilibrium, and the PIC was 0.33, the He 0.41 and Ne 1.70. At the fourth locus A-1027G, the allele A was dominant to G, while the AA was dominant to the remaining genotypes. The frequencies of A and AA were 0.81 and 0.67, respectively. As the immediate 2 loci, the locus A-1027G did not meet the balance of Hardy-Weinberg, and PIC, He and Ne were 0.26, 0.31 and 1.44, respectively.
(3) The subsequent association analysis of these 4 loci with the corresponding reproductive traits demonstrated that the locus A-3684G was absolutely unrelated to the all characters examined. However, the SNP T-3722C showed significant correlation with some traits, such as the live litter size and litter weight at birth, as well litter weight at the weaning of 21 day of homozygote TT and heterozygote TC were higher than those of homozygote CC, respectively. For the locus C-3522G, the homozygote CC and heterozygote CG were lower than homozygote GG in the weaning litter weight. For the locus A-1027G, the homozygote AA and heterozygote AG were found super than homozygote GG in the live birth litter size and birth litter weigh. The bioinformatics analysis employed tfsearch and the other online program for transcriptional factors demonstrated that the 3 SNP sites highly related to the reproductive traits were all in or closely near the putative transcriptional factor binding sites located in the promoter region, while the remaining SNP unrelated to the phenotypes was located beyond any predicted TF binding sites.
Therefore, it can be reasonably concluded as follows:
(1)Swine BMP7 gene promoter region polymorphism was screened and found 39 sites.
(2) The promoter region of porcine BMP7 is rich in the genetic variations, implying a great role in the regulation of this gene as well as the phenotypes related to the reproduction.
(3) Though the further assertion is needed, the loci T-3722C, C-3522G and A-1027G may affect the reproductive traits of sows by modulating the transcription activity of swine BMP7. These polymorphism sites can be expected to develop possible molecular markers for the selection of corresponding traits in the swine breeding.
(1)应用猪毛囊提取的基因组DNA池,通过测序,首次发现启动子区存在39个多态位点。
(2)以猪BMP7基因测序结果为基础,应用PCR-RFLP技术首次检测启动子区4个SNP位点T-3722C、A-3684G、C-3522G和A-1027G。测序结果表明4个SNP位点分别为T/C突变、A/G突变、C/G突变和A/G突变,4个位点都有2个等位基因,3种基因型。遗传变异分析发现,T-3722C位点T是优势等位基因(0.67),优势基因型为TC型(0.48),该位点处于Hardy-Weinberg平衡状态,多态信息含量(PIC)、杂合度(He)、有效等位基因数(Ne)分别为0.34、0.44、1.80,属中度多态。A-3684G位点G是优势等位基因(0.84),优势基因型为GG型(0.76),该位点处于Hardy-Weinberg非平衡状态,PIC、He、Ne分别为0.23、0.27、1.36,属低度多态。C-3522G位点C是优势等位基因(0.71),优势基因型为CG型(0.48),该位点处于Hardy-Weinberg非平衡状态,PIC、He、Ne分别为0.33、0.41、1.70,属中度多态。A-1027G位点A是优势等位基因(0.81),优势基因型为AA型(0.67),该位点处于Hardy-Weinberg平衡状态,PIC、He、Ne分别为0.26、0.31、1.44,属中度多态。
(3)将检测到的4个SNP位点与繁殖性状相关的生产数据进行关联分析。结果显示,A-3684G位点与猪繁殖性状进行关联分析均表现无显著关联(P>0.05)。T-3722C位点与产活仔数、出生窝重及21日龄窝重显著相关(P<0.05);TT和TC型产产活仔数、出生窝重及21日龄窝重均极显著高于CC型。C-3522G位点与21日龄窝重极显著相关(P<0.01),CC和CG型极显著低于GG型。A-1027G位点与产活仔数及出生窝重显著相关(P<0.05),AA和AG型显著高于GG型。应用TFsearch等在线程序对猪BMP7启动子进行分析,预测到3个与繁殖性状显著关联的SNP位点均正好位于或靠近某些重要转录因子结合位点,而与繁殖性状无关联的位点则不在可能的转录因子结合位点区域。
基于以上结果,可得出如下结论:
(1)对猪BMP7基因启动子区域多态位点进行了筛选,发现了39个位点。
(2)猪BMP7基因启动子区域遗传变异性丰富,可能对基因表达乃至表型产生较大的影响。
(3)4个位点中T-3722C、C-3522G和A-1027G 3个位点与猪若干繁殖性状显著关联,它们可能通过改变启动子的转录活性而影响BMP7基因表达及相应的表型。这些多态位点可望作为分子标记用于育种中的辅助选择。
Bone morphogenetic protein 7 is a member of the family of bone morphogenetic proteins. The multifunctional protein also plays an important role in formation of bone and cartilage, and is also involved in the regulation of mammalian reproduction. No reports on the genetic diversity analysis have been found so far in the studies of porcine BMP7 gene, thus the present study took this gene as a target gene for the pig reproductive traits. First, a DNA pool was build using DNA samples from 100 sows of different breeds, and sequenced to screen the putative SNPs occurred in the promoter region. Then after, the PCR-RFLP was employed to genotype the total 255 sows and gilts including 26 Landrace, 147 Largewhite, 82 Doroc at the 4 loci (T-3722C, A-3684G, C-3522G, A-1027G). The main results are as follows:
(1) A total 39 polymorphic loci, including of 3 deletes and 36 signal nucleotide polymorphisms, were pick out for the first time in the studies of this gene.
(2) The genotyping for the 4 variations found reveled that each of these loci occurred in the population studied with the full 3 genotypes. Of 4 sites, T-3722C was dominated by the allele T and genotype TC, with an allele frequency and genotype frequency of 0.67 and 0.48, respectively. The further examination showed that this locus coincided with the Hardy-Weinberg equilibrium, with a polymorphic information content(PIC) ,heterozygosity (He)and effective allele number(Ne) of 0.34,0.44 and 1.80 in the population of interest, respectively. At the locus A-3684G, the dominated allele and genotype were G (0.84) and GG (0.76), respectively. Whereas, this locus was not in the balance of Hardy-Weinberg, and PIC, He and Ne were 0.23, 0.27 and 1.36, respectively. For another locus C-3522G, the dominances were C and CG, with the frequencies of 0.71 and 0.48, respectively. The locus C-3522G was also not consistent to the Hardy-Weinberg equilibrium, and the PIC was 0.33, the He 0.41 and Ne 1.70. At the fourth locus A-1027G, the allele A was dominant to G, while the AA was dominant to the remaining genotypes. The frequencies of A and AA were 0.81 and 0.67, respectively. As the immediate 2 loci, the locus A-1027G did not meet the balance of Hardy-Weinberg, and PIC, He and Ne were 0.26, 0.31 and 1.44, respectively.
(3) The subsequent association analysis of these 4 loci with the corresponding reproductive traits demonstrated that the locus A-3684G was absolutely unrelated to the all characters examined. However, the SNP T-3722C showed significant correlation with some traits, such as the live litter size and litter weight at birth, as well litter weight at the weaning of 21 day of homozygote TT and heterozygote TC were higher than those of homozygote CC, respectively. For the locus C-3522G, the homozygote CC and heterozygote CG were lower than homozygote GG in the weaning litter weight. For the locus A-1027G, the homozygote AA and heterozygote AG were found super than homozygote GG in the live birth litter size and birth litter weigh. The bioinformatics analysis employed tfsearch and the other online program for transcriptional factors demonstrated that the 3 SNP sites highly related to the reproductive traits were all in or closely near the putative transcriptional factor binding sites located in the promoter region, while the remaining SNP unrelated to the phenotypes was located beyond any predicted TF binding sites.
Therefore, it can be reasonably concluded as follows:
(1)Swine BMP7 gene promoter region polymorphism was screened and found 39 sites.
(2) The promoter region of porcine BMP7 is rich in the genetic variations, implying a great role in the regulation of this gene as well as the phenotypes related to the reproduction.
(3) Though the further assertion is needed, the loci T-3722C, C-3522G and A-1027G may affect the reproductive traits of sows by modulating the transcription activity of swine BMP7. These polymorphism sites can be expected to develop possible molecular markers for the selection of corresponding traits in the swine breeding.
引文
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