【作者】 李飞；

【导师】 陈宏；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 本研究采用PCR-SSCP、PCR-RFLP和DNA测序技术,检测了南阳牛、郏县红牛、秦川牛、安格斯牛、哈萨克牛和中国荷斯坦牛6个群体共1181个体的4个基因（NUCB1, NUCB2, GAD1, GAD2）编码区的42个DNA位点的遗传变异情况,并应用遗传学和生物统计学分析软件分析了其遗传结构和遗传多样性,以及对南阳牛、郏县红牛和秦川牛3个群体在上述基因位点多态性与牛生长发育体尺性状进行了关联分析,以检验这些基因的多态对3个黄牛群体生长发育的遗传效应,以期发现对重要经济性状具有显著效应的遗传标记,为中国黄牛的高效选育和分子标记数据库的建立、种质资源保存与利用提供遗传学依据。本研究获得了以下结果：1. NUCB1基因SNP及其与南阳牛、郏县红牛和秦川牛生长性状的关联分析实验研究了6个群体NUCB1基因12个外显子位点。新发现4个SNP（4191 G>C、6677G>A、13404A>C、和13421C>U,与NC₀07316对照）,卡方检验显示在6个群体中,唯独荷斯坦奶牛在NUCB1基因四个外显子突变位点的基因及基因型频率处于Hardy-Weinberg平衡状态（P>0.05）,而其余5个群体均有Hardy-Weinberg不平衡状态,说明奶牛与地方黄牛和安格斯牛选择目标不一样。突变位点与南阳牛、秦川牛、郏县红牛生长性状的关联分析表明,NUCB1基因第2个外显子位点多态性只对南阳牛生长性状有显著影响（P<0.05）,突变纯合子体重和胸围性状显著高于野生型,但其对郏县红牛和秦川牛没有影响（P>0.05）。其它突变位点对牛生长性状没有影响（P>0.05）。在NUCB1基因的第1、3、5、6、8、9、10、11、和12位点处（均包括外显子）,经SNP检测,结果均没有发现有遗传变异现象2.NUCB2基因SNP及其与南阳牛、郏县红牛和秦川牛生长性状的关联分析在NUCB2基因11个位点中,新发现8个SNP（13530A>C、1432 G>C、18052 T>C、23539 A>C、25439 A>T、26108 T>A、27451 G>A和27472 T>C,与NC₀07313对照）。卡方检验显示在6个群体中,唯独荷斯坦奶牛在NUCB2基因8个外显子突变位点的基因及基因型频率处于Hardy-Weinberg平衡状态（P>0.05）,而其余5个群体均有Hardy-Weinberg不平衡状态,说明奶牛与地方黄牛和安格斯牛选择目标不一样。突变位点与南阳牛、秦川牛、郏县红牛生长性状的关联分析表明,NUCB2基因第2个外显子位点多态性对郏县红牛和秦川牛生长性状有显著影响（P<0.05）,NUCB2基因第9个外显子位点多态性对南阳牛、郏县红牛和秦川牛生长性状有显著影响,突变纯合子体重和胸围性状显著大于野生型（P<0.05）,但其它突变位点对牛生长性状没有影响（P>0.05）。在NUCB2基因的第5、8、10和11位点（均包括外显子）,经SNP检测,结果均没有发现有遗传变异现象。3.GAD1基因SNP及其与南阳牛、郏县红牛和秦川牛生长性状的关联分析在GAD1基因16个位点中,检测了第1-5个外显子位点,新发现3个SNP（1802 T>C、5056 C>T、11345 G>T、,与NC 007300对照）。在6个群体中卡方检验显示,唯独荷斯坦奶牛GAD1基因3个外显子突变位点的基因及基因型频率处于Hardy-Weinberg平衡状态（P>0.05）,而其余5个群体均有Hardy-Weinberg不平衡状态（P<0.05）,说明奶牛与地方黄牛和安格斯牛选择目标不一样。突变位点与南阳牛、秦川牛、郏县红牛生长性状的关联分析表明,GAD1基因第3个外显子位点多态性对南阳牛、郏县红牛和秦川牛生长性状均有显著影响（P<0.05）,GAD1基因第3个外显子位点多态性对南阳牛、郏县红牛和秦川牛生长性状有显著影响,突变纯合子体重和胸围性状显著大于野生型（P<0.05）,但其它突变位点对牛生长性状没有影响（P>0.05）。4.GAD2基因SNP及其与南阳牛、郏县红牛和秦川牛生长性状的关联分析在GAD2基因16个位点中,检测了16个外显子位点,新发现4个SNP（1238 G>A、7731C>A、26357 G>T和37887 T>A,与NC 007311对照）。在6个群体中卡方检验显示,唯独荷斯坦牛GAD2基因4个外显子突变位点的基因及基因型频率处于Hardy-Weinberg平衡状态（P>0.05）,而其余5个群体均有Hardy-Weinberg不平衡状态（P<0.05）,说明奶牛与地方黄牛和安格斯牛选择目标不一样。突变位点与南阳牛、秦川牛、郏县红牛生长性状的关联分析表明,GAD2基因第10个外显子位点多态性只对南阳牛生长性状均有显著影响,突变纯合子体重和胸围性状显著低于南阳牛野生型（P<0.05）,但对郏县红牛和秦川牛没有用显著影响（P>0.05）,其它突变位点对牛生长性状没有显著影响（P>0.05）。GAD2基因的第1、3、4、5、7、9、11、12、13、14、15、和16位点（均包括外显子）,经SNP检测,结果均没有发现有遗传变异现象。综上所述,本实验研究创新点概括为以下3点：（1）系统研究了黄牛NUCB1、NUCB2、GAD2基因所有外显子和GAD1基因5个外显子共42个位点多态性,检测到19个SNP,其中17个为首次发现,分析了突变位点的群体遗传特征和与生长性状的关联性。（2）首次揭示了核组蛋白和谷氨酸脱羧酶基因在安格斯牛、哈萨克牛、荷斯坦奶牛、南阳牛、郏县红牛和秦川牛遗传变异的现状,发现奶牛在所检测的4个基因所有突变位点中,其基因和基因型频率均处在Hardy-Weinberg平衡之中,与地方黄牛品种存在明显区别,说明奶牛选择目标与黄牛选择指标不同,其基因频率有明显区别。（3）发现了2个与3个黄牛群体的体重和胸围性状都有关联的多态位点,即NUCB2第9位点和GAD1的第3位点,在南阳牛、郏县红牛和秦川牛群体中,其突变纯合基因型的体重和胸围均大于野生型,这2个SNP位点可用作体重和胸围的分子标记。更多还原

【Abstract】 Genetic variations of energy homeostasis related genes （NUCB2, NUCB1, GAD1, GAD2,） were detected by PCR-RFLP, PCR-SSCP and DNA sequencing techniques in 1036 individuals of seven populations （Nanyang, Qinchuan, Jiaxian Red cattle, Hasak, Angus, and Chinese Holstein）, and association analysis were carried out to evaluate the effects of genotypes of candidate genes on growth traits of three Chinese cattle. The objects were to discovery the hereditary characteristics and to explore molecular markers with significant effects on economic important traits for efficient selection and improvement of Chinese cattle, and to provide genetic information for foundation of molecular marker database, protection and usage of breed resource of Chinese cattle. The results were as follows:1. Relationship between genetic variation of exon2 and exon4 in NUCB1 gene and growth traits in Nanyang and Qinchuan cattleIn this study, polymorphism in the exon 1,2,3,4,5,6,7,8,9,10 11and 12 of bovine NUCB1 gene was detected by PCR-SSCP and DNA sequencing methods in 1181 individuals from six Chinese cattle breeds. The varations of four loci in NUCB1 gene were studied. Four SNPs （4191 G>C,6677 G>A,13404 A>C and 13421 C>U, ref. NC₀07316） were detected in 12 exones for the first time. Two haplotypes （A and B） and three observed genotypes （AA, AB, and BB）. Those nucleotide sequences showed one mutations （G4191C） by comparisons with NC₀07316 （bovine NUCB1 gene DNA sequence）:the NC₀07316:g.4191G>C mutations at the exon2; the NC₀07316:g.4191T>C mutation identified a missense mutation SNP （p.R67T）, namely, Ile （AGG）> Ser （ACG） at position 67. But polymorphism of the NUCB1 gene was shown to not be associated with growth traits in the Nanyang, Jiaxian red and Qinchuan breed（P>0.05）.Two haplotypes （A and B） and three observed genotypes （AA, AB, and BB）. Those nucleotide sequences showed one mutations （G6677A） by comparisons with NC₀07316 （bovine NUCB1 gene DNA sequence）:the NC₀07316:g.6677G>A mutations at the exon4; the NC₀07316:g.6677 G>A mutation identified a missense mutation SNP （p.F67T）, namely, Phle （GCA）> Ser （ACA） at position 401. But polymorphism of the NUCB1 gene was shown to not be associated with growth traits in the Nanyang, Jiaxian red and Qinchuan breed（P>0.05）. 2. Relationship between genetic variation of NUC2 gene and growth traits in three Chinese cattleIn this study, polymorphism in the exon 1,2,3,4,5,6,7,8,9,10 and 11 of bovine NUCB2 gene was detected by PCR-SSCP and DNA sequencing methods in 1811 individuals from six Chinese cattle breeds. The varations of eight loci in NUCB2 gene were studied. Eight SNPs （13530 A>C,1432 G>C,18025 T>C,23539 A>C,25439 A>T,26108 T>A,27451 G>A and 27472 T>C, ref. NC₀07313） were detected in 11 exones for the first time. Two haplotypes （A and B）, three observed genotypes （AA, AB, and BB） and two SNPs （NC₀07313:g.27451G>A, NC₀07313:g.27472T>C） were detected. The frequencies of haplotypes A and B in inland Chinese three breeds were 0.414～0.494 and 0.506～0.586 respectively. The studied Nanyang, Jiaxian red and Qinchuan cattle populations were in Hardy-Weinberg equilibrium at SNPs locus of NUCB2 gene （P>0.05）. Polymorphism of the NUCB2 gene was shown to be associated with growth traits in the three breed. The linkage of two mutant site in the bovine NUCB2 gene had significant effects on body length, body weight, heart girth, and average daily gain at 24 months old （P<0.05）. Results of this study suggest that the NUCB2-gene-specific SNP may be a useful marker for growth traits in future marker-assisted selection programmes in inland Chinese cattle.Two haplotypes （A and B） and three observed genotypes （AA, AB, and BB）. Those nucleotide sequences showed one mutations （G18025C） by comparisons with NC₀07313 （bovine NUCB2 gene DNA sequence）:the NC₀07313:g.18025T>C mutations at the exon 3; the NC₀07313: g.18025T>C mutation identified a missense mutation SNP （p.I34S）, namely, Ile （AUU）> Ser （AGU） at position 34. But polymorphism of the NUCB2 gene was shown to not be associated with growth traits in the Nanyang, Jiaxian red and Qinchuan breed（P<0.05）.3. Relationship between genetic variation of exon3 in GAD1 gene and growth traits in three Chinese cattleIn this study, polymorphism in the exon 1,2,3,4 and 5 of bovine GAD1 gene was detected by PCR-SSCP and DNA sequencing methods in 1811 individuals from six Chinese cattle breeds. The varations of three loci in GAD1 gene were studied. Three SNPs （5056 C>T,11345 T>C, 1802 T>C, ref. NC₀07300） were detected in 5 exones for the first time. Two haplotypes （A and B） and three observed genotypes （AA, AB, and BB）. Those nucleotide sequences showed one mutations （T12345C） by comparisons with NC₀07300 （bovine GADl gene DNA sequence）:the NC₀07300:g.12345T>C mutations at the exon 3; the NC₀07300:g.12345T>C mutation identified a missense mutation SNP （p.F51L）, namely, Phe （TTT）> Leu （CTT） at position 51. Polymorphism of the GAD1 gene was shown to be associated with growth traits in the Nanyang breed. The SNP in the bovine GADl gene had significant effects on body length, body weight, heart girth, and average daily gain at 24 months old （P<0.05）.4. Relationship between genetic variation of exon10 in GAD2 gene and growth traits in Nanyang and Qinchuan cattleIn this study, polymorphism in the exon 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15 and 16 and 5 of bovine GAD2 gene was detected by PCR-SSCP and DNA sequencing methods in 1181 individuals from six Chinese cattle breeds. Four SNPs （1238 G>A、1238 G>A、26357 G>T、37887 T>A ref. NC₀07311） were detected in 16 exones for the first time.Two haplotypes （A and B） and three observed genotypes （AA, AB, and BB）. Those nucleotide sequences showed one mutations （T37887A） by comparisons with NC₀07311 （bovine GAD2 gene DNA sequence）:the NC₀07311:g.37887T>A mutations at the exon 10; the NC₀07311:g.37887T>A mutation identified a missense mutation SNP （p.L1019Q）, namely, Leu （CTG）> Gln （CAG） at position 1019. Polymorphism of the GAD2 gene was shown to only be associated with growth traits in the Nanyang breed. The SNP in the bovine GAD2 gene had significant effects on body weight, and heart girth at 24 months old （P<0.05）.Two haplotypes （A and B） and three observed genotypes （AA, AB, and BB）. Those nucleotide sequences showed one mutations （G1238A） by comparisons with NC 007311 （bovine GAD2 gene DNA sequence）:the NC₀07311:g.12387G>A mutations at the exon 6; the NC 007311:g.1238T>A mutation identified a homonymy mutation SNP （p.K221K）, namely, Lys （CCC）> Lys （CCA） at position 221. Polymorphism of the GAD2 gene was shown to be not associated with growth traits in the Nanyang Jiaxian red and Qinchuan breeds（P>0.05）.更多还原