【作者】 王谋；

【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， 2009， 硕士

【摘要】 本研究采用PCR-SSCP、PCR-RFLP和DNA测序等技术,检测了秦川牛、南阳牛、郏县红牛和中国荷斯坦牛4个品种共818个个体的内脏脂肪素（Visfatin）和视黄醇结合蛋白4（RBP4）基因9个基因座的遗传变异,分析了其遗传结构和遗传多样性。同时利用SPSS（16.0）软件,运用最小二乘拟合线性模型等方法对秦川牛、南阳牛和郏县红牛3个黄牛品种在上述基因座中的多态性与生长性状进行了关联分析,分析了这些基因多态性对黄牛生长发育的遗传效应,以期发现对我国部分牛品种重要经济性状具有显著效应的遗传标记,为高效选育提供遗传学依据。本试验研究获得了以下结果:1. 4个牛品种Visfatin基因多态性分析本试验共检测了Visfatin基因7个基因座（V1～V7）,包含外显子2、4、5、6、8、9和外显子11以及部分内含子。在所研究的7个基因座中,仅V2和V4基因座发现多态性。在秦川牛、南阳牛和郏县红牛中,V2基因座首次在内含子4区域发现长度为35 bp插入突变现象（NW₀01494891:g14438-14439 Ins ACT GGA ATT CTA GTT TAA AAA TTG CTA CTA ATG AA）,但在中国荷斯坦牛中未发生相关突变。在这4个牛品种中,V4基因座首次在内含子5区域发现长度为6 bp缺失突变现象（NW₀01494891:g19768-19773 Del TAA AAA）。同时,该6 bp缺失突变导致了1个DraI内切酶酶切位点的缺失。卡方检验显示:在V2和V4基因座,4个品种均处于Hardy-Weinberg平衡状态。在V2和V4基因座中,秦川牛、南阳牛、郏县红牛和中国荷斯坦牛4个品种的群体遗传多态指数Ne/PIC/He分别为: 1.60/0.30/0.37 , 1.99/0.37/0.50 , 1.90/0.36/0.47 ,1.00/0.00/0.00;1.12/0.10/0.11,1.16/0.13/0.14,1.08/0.07/0.07,1.02/0.02/0.02。基因型分布的卡方独立性检验显示:在V2基因座中除郏县红牛与南阳牛之间差异不显著外,其余品种间均差异极显著（P<0.01）;在V4基因座中除秦川牛与南阳牛、郏县红牛之间差异不显著外,其余品种间均差异显著或极显著（P<0.05或P<0.01）。结果说明,V2基因座在这3个黄牛品种中（秦川牛、南阳牛和郏县红牛）多态信息含量较丰富,选择潜力较大;V4基因座在这4个品种中多态信息含量较贫乏,选择潜力较小。2. Visfatin基因多态性与秦川牛、南阳牛和郏县红牛生长性状的关联分析在牛Visfatin基因V2基因座中检测出WW、WI、II型3种基因型,其中WW型为野生型。在秦川牛的体重、体高、坐骨端宽、胸围方面,在南阳牛的初生重、18月龄体重和日增重方面,在郏县红牛的体高、坐骨端宽和管围方面,WW型均极显著大于II型（P<0.01）。在V4基因座中检测出WW、WD型2种基因型,其中WW型为野生型。在郏县红牛中该基因座不同基因型与其生长性状无显著性影响;在秦川牛的体重和胸围方面以及在南阳牛的初生重、6月龄和12月龄的体重和平均日增重方面,WW型均极显著大于WD型（P<0.01）。结果说明,V2基因座不同基因型与秦川牛、南阳牛和郏县红牛品种体尺和体重存在相关,V4基因座不同基因型与秦川牛和南阳牛品种的体尺和体重存在相关。3. 4个牛品种RBP4基因多态性分析本试验共检测RBP4基因2个基因座（R1和R2）,包含外显子2和3以及部分内含子。在这2个位点上,秦川牛、南阳牛和郏县红牛中均表现多态,而中国荷斯坦牛中均表现为1种带型。首次在R1基因座内含子2区域发现1处SNP位点（NC₀07327:g443C>T）,该处突变导致1个Hin6I内切酶酶切位点的缺失;在R2基因座上内含子2区域发现4 bp缺失突变,外显子3区域发现1处SNP位点（NC₀07327:g3486-3489 Del TCTG;g3571C>G/T）,其中3571C>G/T分别导致RBP4蛋白第3外显子中第15位氨基酸由Pro突变为Arg/Leu,且研究发现这2处突变完全连锁。卡方检验显示:在R1和R2基因座中,4个品种均处于Hardy-Weinberg平衡状态。在R1和R2基因座中,秦川牛、南阳牛、郏县红牛和中国荷斯坦牛的群体遗传多态指数Ne/PIC/He分别为1.63/0.31/0.39,1.87/0.35/0.46,1.90/0.36/0.47,1.00/0.00/0.00;1.51/0.28/0.34,1.66/0.32/0.40,1.52/0.28/0.34,1.00/0.00/0.00。基因型分布的卡方独立性检验显示:在R1基因座中4个牛品种间均差异极显著（P<0.01）;在R2基因座中,除郏县红牛与秦川牛和南阳牛之间差异不显著外,其余品种间均差异极显著（P<0.01）。结果说明,R1和R2基因座在3个黄牛品种中（秦川牛、南阳牛和郏县红牛）多态信息含量较丰富,选择潜力较大。4. RBP4基因多态性与秦川牛、南阳牛和郏县红牛生长性状的关联分析在RBP4基因R1基因座中检测出CC、CT、TT型3种基因型,其中CC型为野生型。在秦川牛的体重、腰角宽、胸围和坐骨端宽方面,TT型均显著大于CC型（P<0.05）。在南阳牛的18月龄坐骨端宽方面,CT型显著大于CC型（P<0.05）;在24月龄体斜长方面,TT型显著大于CT型（P<0.05）。在郏县红牛的体高方面,TT型显著大于CC型（P<0.05）。在R2基因座中检测出CC、CG、CT、GG和GT型5种基因型,其中CC型为野生型。在秦川牛的体重、体高、十字部高和胸围方面,CC型均显著或极显著大于GT和GG型（P<0.05或P<0.01）;在南阳牛的初生重以及6月龄、12月龄、18月龄、24月龄的平均日增重方面,CC型均显著或极显著大于CT和GT型（P<0.05或P<0.01）;在郏县红牛的体高、十字部高方面,CC型均显著或极显著大于CT和GT型（P<0.05或P<0.01）。结果说明,R1和R2基因座不同基因型均与秦川牛、南阳牛和郏县红牛品种的体尺和体重存在相关性。更多还原

【Abstract】 Genetic variations of Visfatin and RBP4 genes were detected by PCR-SSCP, PCR-RFLP and DNA sequencing techniques in 818 individuals from four cattle breeds （Qinchuan, Nanyang, Jiaxian Red cattle and Chinese Holstein）, and their genetic structure and diversity were analyzed; meanwhile, association analysis were carried out by using SPSS software and Least Square Analysis method on growth traits at the candidate genes of the three Chinese cattle breeds （Qinchuan, Nanyang and Jiaxian Red cattle）. The objects were to discovery the hereditary characteristics of these cattle breeds, to explore genetic markers with significant effects on economic important traits for efficient selection, and to provide genetic information for the cattle breeding program. The results were as follows:1. Polymorphisms of Visfatin gene in four breeds of cattleThe variations of seven loci （V1^V7） in Visfatin gene were detected. These loci contained exon2, exon4, exon5, exon6, exon8, exon9, exon11 and partial introns. One 35 bp insertion mutation （ref. NW₀01494891: g14438-14439 Ins ACT GGA ATT CTA GTT TAA AAA TTG CTA CTA ATG AA） was detected at V2 locus in intron 4 for the first time in three cattle populations （Qinchuan, Nanyang, and Jiaxian Red cattle）. This mutation was not found in Chinese Holstein breed. A 6 bp deletion mutation （ref. NW₀01494891: g19768-19773 Del TAA AAA） was detected in intron 5 for the first time at V4 locus in these four cattle breeds. Interestingly, this deletion mutation caused the lost of a DraI cutting site. The results illustrated that V2 locus contained more abundant polymorphic information than V4 locus, and V2 locus has more selection potential.Chi-square test results were as follows: both V2 and V4 locus were in Hardy-Weinberg equilibrium in the four breeds. Genetic diversity indexes （Ne/PIC/He） in Qinchuan, Nanyang, Jiaxian Red cattle and Chinese Holstein were 1.60/0.30/0.37, 1.99/0.37/0.50, 1.90/0.36/0.47, and 1.00/0.00/0.00 respectively at V2 locus and 1.12/0.10/0.11, 1.16/0.13/0.14, 1.08/0.07/0.07 and 1.02/0.02/0.02 respectively at V4 locus. The Chi-square independence tests, which detect the differences between/ among the four breeds, showed that there were significant differences between/among the four cattle populations in genotype distribution （P<0.01）, except the results between Nanyang and Jiaxian Red cattle at V2 locus. At V4 locus, there were significant differences between/among the four cattle breeds in genotype distribution （P<0.05 or P<0.01）, except the results between Qinchuan and Nanyang, as well as Qinchuan and Jiaxian Red cattle breeds.2. Relationships between genetic variation of Visfatin gene and growth traits in Qinchuan, Nanyang and Jiaxian Red cattleThree genotypes （WW, WI and II） were found at V2 locus in Visfatin gene. Genotype WW was the wild genotype. In Qinchuan cattle population, the individuals with genotype WW had significantly greater body weight, withers height, hucklebone width and heart girth than the ones with genotype II （P<0.01）; in Nanyang cattle population, the animals with genotype WW had significantly greater birth weight, 18-month weight and average daily gain than the ones with genotype II （P<0.01）; in Jiaxian Red cattle breed, the individuals with genotype WW owned significant greater withers height, hucklebone width and heart girth than the ones owned genotype II （P<0.01）. Two genotypes （WW and WD） were detected at V4 locus. The genotype WW was the wild genotype. In Qinchuan cattle breed, the individuals with genotype WW had significantly greater body weight and heart girth than the ones with genotype WD （P<0.01）; in Nanyang cattle breed, the animals with genotype WW had significantly greater birth weight, 6-month body weight, 12-month body weight and average daily gain than the ones with genotype WD （P<0.01）; there was no significant influence in Jiaxian Red cattle. The results illustrated that different genotypes has different relationships with growth traits in Qinchuan, Nanyang, and Jiaxian Red cattle at V2 locus; meanwhile, different genotypes has different relationships with growth traits in Qinchuan, and Nanyang cattle at V4 locus.3. Polymorphisms of RBP4 gene in four breeds of cattleTwo loci （R1, R2） were detected at RBP4 gene, which include exon 2, exon 3 and partial introns. Except the Chinese Holstein breed, mutations were detected at the two loci in the other three breeds. One SNP was firstly found in intron 2 at R1 locus （NC₀07327: g443C>T）; meanwhile, this SNP caused the lost of a Hin6I restriction site. A SNP and a 4-bp deletion were found for the first time at R2 locus （NC₀07327: g443C>T; g 3486-3489 Del TCTG; g3571C>G/T）. This SNP （g3571C>G/T） caused the animo acid changed from Pro to Arg/Leu respectively. These two mutations were complete linkage.Chi-square test results were as follows: R1 and R2 locus were both in Hardy-Weinberg equilibrium in the four breeds. Genetic diversity indexes of Ne/PIC/He in Qinchuan, Nanyang, Jiaxian Red cattle and Chinese Holstein were 1.63/0.31/0.39, 1.87/0.35/0.46, 1.90/0.36/0.47, and 1.00/0.00/0.00 respectively at R1 locus and 1.51/0.28/0.34, 1.66/0.32/0.40, 1.52/0.28/0.34 and 1.00/0.00/0.00 respectively at R2 locus. The Chi-square independence tests showed that there were significant differences between/among the four cattle populations in genotype distribution （P<0.01） at R1 and R2 locus except the results between Qinchuan and Jiaxian Red breed, as well as Nanyang and Jiaxian Red cattle. The results illustrated that both the R1 and R2 loci owned abundant polymorphic information, and both of them has strong selection potential.4. Relationships between genetic variation of RBP4 gene and growth traits in Qinchuan, Nanyang and Jiaxian Red cattleThree genotypes （CC, CT and TT） were found at R1 locus in RBP4 gene. The CC genotype was the wild genotype. In Qinchuan cattle breed, the individuals with genotype TT had greater body weight, hip width, heart girth and hucklebone width than the ones with CC genotype （P<0.05）; in Nanyang cattle breed, the animals with genotype CT had greater 18-month hucklebone width than the ones with genotype CC, and the animals with genotype TT had longer 24-month body length than the ones with genotype CT （P<0.05）. In Jiaxian Red cattle breed, the ones with genotype TT had greater withers height than the ones with genotype CC （P<0.05）.CC, CG, CT, GG and GT genotypes were found at R2 locus. Genotype CC was the wild genotype. In Qinchuan cattle breed, the individuals with genotype CC had greater body weight, withers height, height at hip cross and heart girth than the individuals with genotype GT or GG （P<0.05 or P<0.01）. In Nanyang cattle breed, the cattle with genotype CC had greater birth weight, 6-month, 12-month, 18-month and 24-month average daily gain than the ones with genotype CT or GT （P<0.05 or P<0.01）. In Jiaxian Red cattle breed, the animals with genotype CC had greater withers height and height at hip cross than the ones with genotype CT or GT （P<0.05 or P<0.01）. The results illustrated that different genotypes has different relationships with growth traits in Qinchuan, Nanyang, and Jiaxian Red cattle at both R1 and R2 locus.更多还原