STAT1、STAT5A基因多态性与中国荷斯坦奶牛产奶性状的相关性研究
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摘要
本试验以199头中国荷斯坦奶牛为研究对象,采用PCR-SSCP的方法,系统的研究了STAT1(GeneID:510814)基因内含子1,4,10,11,18,19,25及3′UTR等十个位点与STAT5A(GeneID: AJ237937)基因外显子7~12,内含子14共17个位点的多态性,并将其与第一至三个胎次的产奶性状进行了关联分析。目的在于探索STAT1、STAT5A基因对中国荷斯坦奶牛产奶性状的影响,为今后良种奶牛的选育提供一定的理论依据。研究结果如下:
在STAT1基因中,在内含子11 P1,P2及3′UTR 3个位点发现了多态性,其余位点在所研究的群体中未发现多态性;在STAT5A基因中,只在内含子9和内含子14两处发现多态,其余位点均没有多态性。各多态位点的突变情况及不同基因型与产奶性状的相关性如下所示:
1. STAT1基因内含子11 P1基因多态性及其与产奶性状的相关性
以中国荷斯坦奶牛基因组DNA为模板,用自行设计的引物P1对STAT1基因的内含子11进行PCR扩增,产物长度为369bp。经聚丙烯酰胺凝胶电泳后发现,该位点存在3种基因型,分别命名为AA,AB,BB;基因型频率分别为0.442,0.462,0.096。经测序发现,突变产生在28739碱基处,为G转换为A。3种基因型与产奶性状的关联分析表明:BB基因型个体第一、二胎次乳蛋白率均显著高于AA型和AB型个体(P<0.05);3种基因型对各胎次产奶量及乳脂率均没有显著影响(P>0.05),但呈现出BB>AB>AA的趋势。
2. STAT1基因内含子11 P2基因多态性及其与产奶性状的相关性
由引物P2扩增出的片段,经聚丙烯酰胺凝胶电泳分型发现,在该扩增片段存在三种基因型,分别命名为CC,CD,DD,基因型频率分别为0.331,0.196,0.473。经测序发现,3种基因型由28873碱基处发生G颠换为C而产生。3种基因型与产奶性状的关联分析表明:DD型个体的第三胎次的产奶量显著高于CC型个体(P<0.05),DD型个体的第二胎次的乳蛋白率显著高于CC和CD型个体(P<0.05),3种基因型对各胎次乳脂率均没有显著影响(P>0.05)。
3. STAT1基因3′UTR基因多态性及其与产奶性状的相关性
本试验对3′UTR不同位置自行设计了3对引物,结果仅在引物1的扩增片段中发现多态性。该位点存在3种基因型,分别命名为TT,CT,CC,基因型频率分别为0.437,0.116,0.447。测序结果表明,突变发生在141930碱基处,为C转换T。3种基因型与产奶性状的关联分析表明:基因型CC,CT个体的第一、二胎次产奶量均显著高于TT型个体(P<0.05);CC型个体的第一、三胎次乳蛋白率均显著高于TT型个体(P<0.05)。三种基因型对三个胎次乳脂率的影响差异均不显著(P>0.05),但呈现出CC>CT>TT的趋势。
4. STAT5A基因内含子9基因多态性及其与产奶性状的相关性
用自行设计的引物对STAT5A基因外显子10进行扩增,经聚丙烯酰胺凝胶电泳分型发现了三种基因型,分别命名为GG,AA ,GA,基因型频率分别为0.112,0.138,0.75。经测序后序列比对发现,突变产生在内含子9,为9501碱基处发生G转换为A的突变。与产奶性状的关联分析表明:AA型个体的第三胎次产奶量极显著高于GA和GG型个体(P<0.01) ,GG型个体第二、三胎次乳蛋白率显著高于AA和GA型个体(P<0.05);GG,GA型个体第一、三胎次乳脂率显著高于AA型个体(P<0.05)。
综上所述,STAT1,STAT5A基因对中国荷斯坦奶牛的产奶性状的影响均达到了显著水平,因此可以将这2个基因作为荷斯坦奶牛分子标记辅助选择的候选基因。
The polymorphisms of seventeen sites which were intron 1,4,10,11,18,19,25, and 3′UTR of STAT1gene(GeneID:510814)and exon7-12, intron 14 of STAT5A gene(GeneID: AJ237937)were detected in 199 Chinese Holstein cattle with PCR-SSCP , and the association between the polymorphisms and milk production traits of three birth orders were analyzed. The aim was to choose perfect markers which can provide theoretical base for breeding in dairy cattle. The results were as follows:
Only three sites existed polymorphism in STAT1 gene and two sites in STAT5A gene, which were intron11 (P1and P2), 3′UTR of STAT1 gene and intron9, 14 of STAT5A gene. The analysis of association between different genotypes and milk traits were showed as follows:
1. The association between polymorphism of intron11 (P1) of STAT1 gene and milk production traits
Primer designed by primer5.0 were used to amplify the DNA of ChineseHolstein cattle, the length of product was 369 bp. Three genotypes AA,AB andBB were identified after PCR-SSCP, the frequency of each genotype was: 0.442,0.462,0.096.The sequencing results showed that the mutation was chage of G→A in position 28739.the analysis between different genotypes and milk production traits showed that : cows with genotype BB had significantly higher protein percent (P%) in the first and second birth orders than that of genotype AA and AB(P<0.05); there were no significant differences among three genotypes on 305 days milk yield, and fat percent (F%) of all birth orders, while the tendency of BB>AB>AA was showed.
2. The association between polymorphism of intron11 (P2) of STAT1 gene and milk production traits
The product amplified by primer P2 was analyzed by PCR-SSCP. There were three genotypes: CC, CD and DD, the frequency of each genotype was: 0.331, 0.196, 0.473. The sequencing results showed that three genotypes were caused by the mutation of G→C in position 28873. the analysis between different genotypes and milk production traits showed that: cows with genotype DD had significantly higher milk yield in the third birth order than that of genotype CC (P<0.05), cows with genotype DD had significantly higher protein percent (P%) in the second birth order than that of genotype CC,CD (P<0.05), there were no significant differences among three genotypes on fat percent (F%) of all birth orders(P>0.05).
3. The association between polymorphism of 3′UTR of STAT1 gene and milk production traits
Three primers were designed to amplify different segments in 3′UTR, while only one segment was found polymorphism. There were three genotypes: TT, CT and CC, the frequency of each genotype was: 0.437, 0.116, 0.447. The sequencing results showed that the mutation was C→T in position 141930. the analysis between different genotypes and milk production traits showed that: Cows with genotype CC or TT had significantly higher 305 days milk yield in the first and second birth orders; and cows with genotype CC had significantly higher protein percent (P%) than genotype TT in the first and third birth orders; there were no significant differences among three genotypes on fat percent (F%).
4. The association between polymorphism of intron9 of STAT5A gene and milk production traits
Exon 10 was amplified by one primer, the segment was analyzed by PCR-SSCP, three genotypes GG, AA and GA were found, and the frequency of each genotype was: 0.112, 0.138, and 0.75. The sequencing results showed that the mutation was in intron 9, which were G→A in position 9501. the analysis between different genotypes and milk production traits showed that : Cows with genotype AA had significantly higher 305 days milk yield than genotype GA and GG in the third birth orders(P<0.01), Cows with genotype GG had significantly higher protein percent (P%) than genotype AA and GA in the second and third birth orders(P<0.05), Cows with genotype GG and GA had significantly higher fat percent (F%) than genotype AA in the first and third birth orders(P<0.05).
We could conclude that the effect of STAT1 gene and STAT5A gene on milk trait was obviously, so these two genes could be used as candidate genes for MAS of Chinese Holstein cattle.
在STAT1基因中,在内含子11 P1,P2及3′UTR 3个位点发现了多态性,其余位点在所研究的群体中未发现多态性;在STAT5A基因中,只在内含子9和内含子14两处发现多态,其余位点均没有多态性。各多态位点的突变情况及不同基因型与产奶性状的相关性如下所示:
1. STAT1基因内含子11 P1基因多态性及其与产奶性状的相关性
以中国荷斯坦奶牛基因组DNA为模板,用自行设计的引物P1对STAT1基因的内含子11进行PCR扩增,产物长度为369bp。经聚丙烯酰胺凝胶电泳后发现,该位点存在3种基因型,分别命名为AA,AB,BB;基因型频率分别为0.442,0.462,0.096。经测序发现,突变产生在28739碱基处,为G转换为A。3种基因型与产奶性状的关联分析表明:BB基因型个体第一、二胎次乳蛋白率均显著高于AA型和AB型个体(P<0.05);3种基因型对各胎次产奶量及乳脂率均没有显著影响(P>0.05),但呈现出BB>AB>AA的趋势。
2. STAT1基因内含子11 P2基因多态性及其与产奶性状的相关性
由引物P2扩增出的片段,经聚丙烯酰胺凝胶电泳分型发现,在该扩增片段存在三种基因型,分别命名为CC,CD,DD,基因型频率分别为0.331,0.196,0.473。经测序发现,3种基因型由28873碱基处发生G颠换为C而产生。3种基因型与产奶性状的关联分析表明:DD型个体的第三胎次的产奶量显著高于CC型个体(P<0.05),DD型个体的第二胎次的乳蛋白率显著高于CC和CD型个体(P<0.05),3种基因型对各胎次乳脂率均没有显著影响(P>0.05)。
3. STAT1基因3′UTR基因多态性及其与产奶性状的相关性
本试验对3′UTR不同位置自行设计了3对引物,结果仅在引物1的扩增片段中发现多态性。该位点存在3种基因型,分别命名为TT,CT,CC,基因型频率分别为0.437,0.116,0.447。测序结果表明,突变发生在141930碱基处,为C转换T。3种基因型与产奶性状的关联分析表明:基因型CC,CT个体的第一、二胎次产奶量均显著高于TT型个体(P<0.05);CC型个体的第一、三胎次乳蛋白率均显著高于TT型个体(P<0.05)。三种基因型对三个胎次乳脂率的影响差异均不显著(P>0.05),但呈现出CC>CT>TT的趋势。
4. STAT5A基因内含子9基因多态性及其与产奶性状的相关性
用自行设计的引物对STAT5A基因外显子10进行扩增,经聚丙烯酰胺凝胶电泳分型发现了三种基因型,分别命名为GG,AA ,GA,基因型频率分别为0.112,0.138,0.75。经测序后序列比对发现,突变产生在内含子9,为9501碱基处发生G转换为A的突变。与产奶性状的关联分析表明:AA型个体的第三胎次产奶量极显著高于GA和GG型个体(P<0.01) ,GG型个体第二、三胎次乳蛋白率显著高于AA和GA型个体(P<0.05);GG,GA型个体第一、三胎次乳脂率显著高于AA型个体(P<0.05)。
综上所述,STAT1,STAT5A基因对中国荷斯坦奶牛的产奶性状的影响均达到了显著水平,因此可以将这2个基因作为荷斯坦奶牛分子标记辅助选择的候选基因。
The polymorphisms of seventeen sites which were intron 1,4,10,11,18,19,25, and 3′UTR of STAT1gene(GeneID:510814)and exon7-12, intron 14 of STAT5A gene(GeneID: AJ237937)were detected in 199 Chinese Holstein cattle with PCR-SSCP , and the association between the polymorphisms and milk production traits of three birth orders were analyzed. The aim was to choose perfect markers which can provide theoretical base for breeding in dairy cattle. The results were as follows:
Only three sites existed polymorphism in STAT1 gene and two sites in STAT5A gene, which were intron11 (P1and P2), 3′UTR of STAT1 gene and intron9, 14 of STAT5A gene. The analysis of association between different genotypes and milk traits were showed as follows:
1. The association between polymorphism of intron11 (P1) of STAT1 gene and milk production traits
Primer designed by primer5.0 were used to amplify the DNA of ChineseHolstein cattle, the length of product was 369 bp. Three genotypes AA,AB andBB were identified after PCR-SSCP, the frequency of each genotype was: 0.442,0.462,0.096.The sequencing results showed that the mutation was chage of G→A in position 28739.the analysis between different genotypes and milk production traits showed that : cows with genotype BB had significantly higher protein percent (P%) in the first and second birth orders than that of genotype AA and AB(P<0.05); there were no significant differences among three genotypes on 305 days milk yield, and fat percent (F%) of all birth orders, while the tendency of BB>AB>AA was showed.
2. The association between polymorphism of intron11 (P2) of STAT1 gene and milk production traits
The product amplified by primer P2 was analyzed by PCR-SSCP. There were three genotypes: CC, CD and DD, the frequency of each genotype was: 0.331, 0.196, 0.473. The sequencing results showed that three genotypes were caused by the mutation of G→C in position 28873. the analysis between different genotypes and milk production traits showed that: cows with genotype DD had significantly higher milk yield in the third birth order than that of genotype CC (P<0.05), cows with genotype DD had significantly higher protein percent (P%) in the second birth order than that of genotype CC,CD (P<0.05), there were no significant differences among three genotypes on fat percent (F%) of all birth orders(P>0.05).
3. The association between polymorphism of 3′UTR of STAT1 gene and milk production traits
Three primers were designed to amplify different segments in 3′UTR, while only one segment was found polymorphism. There were three genotypes: TT, CT and CC, the frequency of each genotype was: 0.437, 0.116, 0.447. The sequencing results showed that the mutation was C→T in position 141930. the analysis between different genotypes and milk production traits showed that: Cows with genotype CC or TT had significantly higher 305 days milk yield in the first and second birth orders; and cows with genotype CC had significantly higher protein percent (P%) than genotype TT in the first and third birth orders; there were no significant differences among three genotypes on fat percent (F%).
4. The association between polymorphism of intron9 of STAT5A gene and milk production traits
Exon 10 was amplified by one primer, the segment was analyzed by PCR-SSCP, three genotypes GG, AA and GA were found, and the frequency of each genotype was: 0.112, 0.138, and 0.75. The sequencing results showed that the mutation was in intron 9, which were G→A in position 9501. the analysis between different genotypes and milk production traits showed that : Cows with genotype AA had significantly higher 305 days milk yield than genotype GA and GG in the third birth orders(P<0.01), Cows with genotype GG had significantly higher protein percent (P%) than genotype AA and GA in the second and third birth orders(P<0.05), Cows with genotype GG and GA had significantly higher fat percent (F%) than genotype AA in the first and third birth orders(P<0.05).
We could conclude that the effect of STAT1 gene and STAT5A gene on milk trait was obviously, so these two genes could be used as candidate genes for MAS of Chinese Holstein cattle.
引文
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