影响荷斯坦母牛产奶性状候选基因的研究
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摘要
本研究以荷斯坦母牛为试验材料,采用聚合酶链式反应(PCR)、聚合酶链式反应-限制性片段长度多态(CAPS)、单链构象多态(SSCP)、序列分析和基因克隆等方法研究PPARGC1A, FASN和STAT5A候选基因的遗传变异,并分析这些基因对荷斯坦母牛5个产奶性状(305 d产奶量、蛋白率、乳脂率、乳糖率、干物质率)的影响,寻找影响荷斯坦母牛产奶性状的主效基因或有较大关联的基因型或遗传标记,为奶牛产奶性状的标记辅助选择提供科学依据。主要研究结果如下:
1) PPARGC1A基因DNA序列中存在C85330T、A103592C、2632delCTTT和T84064C四个突变位点。C85330T突变位点经HaeⅢ酶切产生三种基因型CC、CT和TT,其频率分别为0.621、0.326和0.053;A103592C突变位点经NheⅠ酶切产生三种基因型AA、AC和CC,基因型频率分别为0.430、0.444、0.126;2632delCTTT突变位点经SSCP检测发现三种基因型EE、EF、FF,其频率分别为0.742、0.237、0.021;T84064C突变位点经SSCP检测发现三种基因型GG、GH、HH,其基因型频率分别为0.770、0.218、0.012。χ2检验表明所检测到的四个多态位点均处于Hardy-Weinberg平衡。关联分析显示,对于C85330T突变位点,TT和CT基因型乳脂率最小二乘均值显著高于CC基因型所对应的值(P<0.05),TT和CT基因型乳脂率最小二乘均值差异不显著(P>0.05);TT是提高乳脂率的最有利基因型,CC是最不利基因型;对于A103592C突变位点,AA和AC基因型蛋白率最小二乘均值显著高于CC基因型所对应的值(P<0.05),AA和AC基因型蛋白率最小二乘均值差异不显著(P>0.05);AA是提高蛋白率的最有利基因型,CC是最不利基因型。对于2632delCTTT和T84064C突变位点,基因型之间5个产奶性状最小二乘均值差异均不显著(P>0.05)。
2) FASN基因DNA序列中存在G15028A和G16923A两个突变位点。G15028A突变位点经SSCP检测发现三种基因型AA、AB、BB,基因型频率分别为0.757、0.233、0.010;G16923A突变位点经SSCP检测发现三种基因型CC、CD、DD,基因型频率分别为0.489、0.425、0.086,且该突变导致丙氨酸变为苏氨酸。χ2检验表明该两个多态位点均处于Hardy-Weinberg平衡。关联分析结果初步显示荷斯坦母牛FASN基因G16923A多态位点的等位基因D与荷斯坦母牛较高乳脂率显著相关(P<0.05),D等位基因是提高奶牛乳脂率的一个潜在的DNA标记。
3) STAT5A基因DNA序列中存在A14217G和17266indelCCT两个突变位点。A14217G突变位点经MspA1Ⅰ酶切产生三种基因型AA、AG和GG,其基因型频率分别为0.252、0.486、0.262;17266indelCCT突变位点经SSCP检测发现三种基因型CC、CT、TT,基因型频率分别为0.751、0.234、0.015。χ2检验表明该两个多态位点均处于Hardy-Weinberg平衡。关联分析表明,AG和GG基因型蛋白率最小二乘均值显著高于AA基因型所对应的值(P<0.05),GG和AG基因型蛋白率最小二乘均值差异不显著(P>0.05);GG是提高蛋白率的最有利基因型,AA是最不利基因型。TT和CT基因型305 d产奶量最小二乘均值显著高于CC基因型所对应的值(P<0.05),TT和CT基因型305 d产奶量最小二乘均值差异不显著(P>0.05);TT是提高305 d产奶量的最有利基因型,CC是最不利基因型;CC和CT基因型乳脂率最小二乘均值显著高于TT基因型所对应的值(P<0.05),CC和CT基因型乳脂率最小二乘均值差异不显著(P>0.05);CC是提高乳脂率的最有利基因型,TT是最不利基因型。
To detect the polymorphisms of PPARGC1A,FASN and STAT5A gene in Holstein cows, CAPS (PCR-RFLP), PCR-PAGE, PCR-SSCP, sequence analysis and gene cloning were used. The relation between the polymorphisms and milk production traits (milk yield at 305d, protein percentage, fat percentage, lactose percentage and dry matter percentage) was also studied to provide a scientific basis for marker-assisted selection of milk production traits in cows. The results were as follows.
1) The products amplified by primers P1, P2, P16 and P18 displayed polymorphisms in PPARGC1A gene. For primer P1, three genotypes (CC, CT and TT) were detected at C85330T site in Holstein cows by digesting the PCR products with restriction endonuclease HaeⅢ, and the frequency of CC/CT/TT was 0.621/0.326/0.053, respectively. Least squares mean of fat percentage for TT or CT was significantly higher than that for CC(P<0.05), the difference of the least squares mean for fat percentage was not significant between TT and CT (P>0.05).For primer P2, three genotypes (AA, AC and CC) were detected at A103592C site in Holstein cows by digesting the PCR products with restriction endonuclease NheⅠ, and the frequency of AA/AC/CC was 0.430/0.444/0.126, respectively. Least squares mean of protein percentage for AA or AC was significantly higher than that for CC (P<0.05), the difference of the least squares mean for protein percentage was not significant between AA and AC (P>0.05). For 2632delCTTT mutation site, three genotypes (EE, EF and FF) were detected in Holstein cows, and the frequency of EE/EF/FF was 0.742/0.237/0.021, respectively. The differences of the least squares means for the five milk production traits between EE, EF and FF were not significant (P>0.05). For primer P18, three genotypes (GG, GH and HH) were detected at T84064C site in Holstein cows, and the frequency of GG/GH/HH was 0.770/0.218/0.012, respectively. The differences of the least squares means for the five milk production traits between GG, GH and HH were not significant (P>0.05).
2) Only the products amplified by primers P3 and P8 displayed polymorphisms. For P3, three genotypes (AA, AB and BB) were detected at G15028A site in Holstein cows, and the frequency of AA/AB/BB was 0.757/0.233/0.010, respectively. For P8, three genotypes (CC, CD and DD) were detected at G16923A site in Holstein cows, and the frequency of CC/CD/DD was 0.489/0.425/0.086, respectively. The two sites were all in Hardy-Weinberg equilibrium. Association analysis showed that the DD genotype of g.16923G>A was associated with a significant increase in fat percentage (P<0.05).
3) The products amplified by primers P1 and P2 displayed polymorphisms. For P1, three genotypes (AA, AG and GG) were detected at G14217A site in Holstein cows, and the frequency of AA/AG/GG was 0.252/0.486/0.262 respectively. For 17266indelCCT mutation site, three genotypes (CC, CT and TT) were detected in Holstein cows, and the frequency of CC/CT/TT was 0.751/0.234/0.015, respectively. The two sites were all in Hardy-Weinberg equilibrium. Association analysis showed that, for P1, the AG and GG genotype were associated with a significant increase in protein percentage (P<0.05). However, for P2, the TT and CT genotype were associated with a significant increase in milk yield at 305 d (P<0.05), and the CC and CT genotype were associated with a significant increase in fat percentage (P<0.05).
1) PPARGC1A基因DNA序列中存在C85330T、A103592C、2632delCTTT和T84064C四个突变位点。C85330T突变位点经HaeⅢ酶切产生三种基因型CC、CT和TT,其频率分别为0.621、0.326和0.053;A103592C突变位点经NheⅠ酶切产生三种基因型AA、AC和CC,基因型频率分别为0.430、0.444、0.126;2632delCTTT突变位点经SSCP检测发现三种基因型EE、EF、FF,其频率分别为0.742、0.237、0.021;T84064C突变位点经SSCP检测发现三种基因型GG、GH、HH,其基因型频率分别为0.770、0.218、0.012。χ2检验表明所检测到的四个多态位点均处于Hardy-Weinberg平衡。关联分析显示,对于C85330T突变位点,TT和CT基因型乳脂率最小二乘均值显著高于CC基因型所对应的值(P<0.05),TT和CT基因型乳脂率最小二乘均值差异不显著(P>0.05);TT是提高乳脂率的最有利基因型,CC是最不利基因型;对于A103592C突变位点,AA和AC基因型蛋白率最小二乘均值显著高于CC基因型所对应的值(P<0.05),AA和AC基因型蛋白率最小二乘均值差异不显著(P>0.05);AA是提高蛋白率的最有利基因型,CC是最不利基因型。对于2632delCTTT和T84064C突变位点,基因型之间5个产奶性状最小二乘均值差异均不显著(P>0.05)。
2) FASN基因DNA序列中存在G15028A和G16923A两个突变位点。G15028A突变位点经SSCP检测发现三种基因型AA、AB、BB,基因型频率分别为0.757、0.233、0.010;G16923A突变位点经SSCP检测发现三种基因型CC、CD、DD,基因型频率分别为0.489、0.425、0.086,且该突变导致丙氨酸变为苏氨酸。χ2检验表明该两个多态位点均处于Hardy-Weinberg平衡。关联分析结果初步显示荷斯坦母牛FASN基因G16923A多态位点的等位基因D与荷斯坦母牛较高乳脂率显著相关(P<0.05),D等位基因是提高奶牛乳脂率的一个潜在的DNA标记。
3) STAT5A基因DNA序列中存在A14217G和17266indelCCT两个突变位点。A14217G突变位点经MspA1Ⅰ酶切产生三种基因型AA、AG和GG,其基因型频率分别为0.252、0.486、0.262;17266indelCCT突变位点经SSCP检测发现三种基因型CC、CT、TT,基因型频率分别为0.751、0.234、0.015。χ2检验表明该两个多态位点均处于Hardy-Weinberg平衡。关联分析表明,AG和GG基因型蛋白率最小二乘均值显著高于AA基因型所对应的值(P<0.05),GG和AG基因型蛋白率最小二乘均值差异不显著(P>0.05);GG是提高蛋白率的最有利基因型,AA是最不利基因型。TT和CT基因型305 d产奶量最小二乘均值显著高于CC基因型所对应的值(P<0.05),TT和CT基因型305 d产奶量最小二乘均值差异不显著(P>0.05);TT是提高305 d产奶量的最有利基因型,CC是最不利基因型;CC和CT基因型乳脂率最小二乘均值显著高于TT基因型所对应的值(P<0.05),CC和CT基因型乳脂率最小二乘均值差异不显著(P>0.05);CC是提高乳脂率的最有利基因型,TT是最不利基因型。
To detect the polymorphisms of PPARGC1A,FASN and STAT5A gene in Holstein cows, CAPS (PCR-RFLP), PCR-PAGE, PCR-SSCP, sequence analysis and gene cloning were used. The relation between the polymorphisms and milk production traits (milk yield at 305d, protein percentage, fat percentage, lactose percentage and dry matter percentage) was also studied to provide a scientific basis for marker-assisted selection of milk production traits in cows. The results were as follows.
1) The products amplified by primers P1, P2, P16 and P18 displayed polymorphisms in PPARGC1A gene. For primer P1, three genotypes (CC, CT and TT) were detected at C85330T site in Holstein cows by digesting the PCR products with restriction endonuclease HaeⅢ, and the frequency of CC/CT/TT was 0.621/0.326/0.053, respectively. Least squares mean of fat percentage for TT or CT was significantly higher than that for CC(P<0.05), the difference of the least squares mean for fat percentage was not significant between TT and CT (P>0.05).For primer P2, three genotypes (AA, AC and CC) were detected at A103592C site in Holstein cows by digesting the PCR products with restriction endonuclease NheⅠ, and the frequency of AA/AC/CC was 0.430/0.444/0.126, respectively. Least squares mean of protein percentage for AA or AC was significantly higher than that for CC (P<0.05), the difference of the least squares mean for protein percentage was not significant between AA and AC (P>0.05). For 2632delCTTT mutation site, three genotypes (EE, EF and FF) were detected in Holstein cows, and the frequency of EE/EF/FF was 0.742/0.237/0.021, respectively. The differences of the least squares means for the five milk production traits between EE, EF and FF were not significant (P>0.05). For primer P18, three genotypes (GG, GH and HH) were detected at T84064C site in Holstein cows, and the frequency of GG/GH/HH was 0.770/0.218/0.012, respectively. The differences of the least squares means for the five milk production traits between GG, GH and HH were not significant (P>0.05).
2) Only the products amplified by primers P3 and P8 displayed polymorphisms. For P3, three genotypes (AA, AB and BB) were detected at G15028A site in Holstein cows, and the frequency of AA/AB/BB was 0.757/0.233/0.010, respectively. For P8, three genotypes (CC, CD and DD) were detected at G16923A site in Holstein cows, and the frequency of CC/CD/DD was 0.489/0.425/0.086, respectively. The two sites were all in Hardy-Weinberg equilibrium. Association analysis showed that the DD genotype of g.16923G>A was associated with a significant increase in fat percentage (P<0.05).
3) The products amplified by primers P1 and P2 displayed polymorphisms. For P1, three genotypes (AA, AG and GG) were detected at G14217A site in Holstein cows, and the frequency of AA/AG/GG was 0.252/0.486/0.262 respectively. For 17266indelCCT mutation site, three genotypes (CC, CT and TT) were detected in Holstein cows, and the frequency of CC/CT/TT was 0.751/0.234/0.015, respectively. The two sites were all in Hardy-Weinberg equilibrium. Association analysis showed that, for P1, the AG and GG genotype were associated with a significant increase in protein percentage (P<0.05). However, for P2, the TT and CT genotype were associated with a significant increase in milk yield at 305 d (P<0.05), and the CC and CT genotype were associated with a significant increase in fat percentage (P<0.05).
引文
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