【作者】 樊月圆；

【导师】 昝林森；

【作者基本信息】 西北农林科技大学 ， 动物学， 2010， 博士

【摘要】 本研究以秦川牛、南阳牛、鲁西牛、郏县红牛、夏南牛以及安格斯牛6个肉用品种共计717个个体为实验动物,利用PCR-SSCP、DNA测序技术及DNA序列分析技术,研究了影响牛脂肪代谢的4个基因(PPARα、PPARδ、PPARγ以及PLIN)的遗传变异,分析了其遗传结构和遗传多样性。并对108头秦川牛在上述位点的多样性与其胴体、肉质性状进行了相关分析,同时利用生物信息学分析了部分分子标记的作用机理,以期筛选出对肉牛重要经济性状具有显著效应的功能基因或分子标记,为肉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。本研究主要获得了以下结果: 1. PPARα基因多态性及其与秦川牛胴体、肉质性状的相关分析采用PCR-SSCP及DNA测序方法,共研究了6个群体PPARα基因的8个基因座,包括PPARα基因的7个外显子及第2内含子。发现了6个新的SNP位点。在PPARα基因的第2内含子检测到3个SNP位点,分别为2666 C>G,2678 T>C和2706 T>C的突变,在该基因座,D等位基因占主导地位,秦川牛、南阳牛、郏县红牛、鲁西牛、夏南牛和安格斯牛等位基因D的频率分别为,0.571、0.404、0.419、0.417、0.422和0.386;PIC值分别为0.421、0.416、0.434、0.447、0.438和0.413,均为中度多态,遗传多态性丰富。该位点与眼肌面积极显著相关(P<0.01),表现为CC基因型个体眼肌面积极显著高于CD基因型个体(P<0.01)。PCR-SSCP检测到在第5外显子的138bp处存在一个T>C的突变,该突变发生在第5外显子和第5内含子的交界处。未引起氨基酸的变化。秦川牛和夏南牛群体处于Hardy-Weinberg不平衡状态(P<0.05),南阳牛、郏县红牛、安格斯和鲁西牛群体处于Hardy-Weinberg平衡状态(P>0.05)。同时这6个牛群体均属于中度多态,遗传多态性丰富程度从大到小依次为秦川牛、郏县红牛、安格斯牛、鲁西牛、夏南牛和南阳牛。138T>C位点与秦川牛的眼肌面积、背膘厚和系水力指标具有相关性。184 C>T突变发生于PPARα基因的第7外显子上,在该位点,秦川牛和安格斯这2个群体处于Hardy-Weinberg非平衡状态(P<0.05),南阳牛、郏县红牛、夏南牛和鲁西牛群体处于Hardy-Weinberg平衡状态(P>0.05)。遗传学指标结果显示:秦川牛、郏县红牛、安格斯、鲁西牛和夏南牛群体属于低度多态,南阳牛属于中度多态。该位点与秦川牛背膘厚和胴体长显著相关(P<0.05)。260 C>T突变发生在PPARα基因的第8外显子上,在南阳牛、秦川牛、郏县红牛、安格斯、鲁西牛和夏南牛中,等位基因G/H的频率分别为0.925/0.075、0.864/0.136、0.791/0.209、0.828/0.172、0.835/0.165和0.853/0.147。在南阳牛群体中未检测到基因型HH。该位点GG基因型为优势基因型,等位基因G为优势等位基因。南阳牛处于Hardy-Weinberg平衡状态(P>0.05),秦川牛、郏县红牛、安格斯、鲁西牛和夏南牛5个群体均处于Hardy-Weinberg不平衡状态(P<0.05)。郏县红牛属于中度多态,南阳牛、秦川牛、安格斯、鲁西牛和夏南牛这5个群体均属于低度多态。260 C>T位点与系水力指标显著相关(P<0.05),在秦川牛群体中,GG基因型个体在系水力性状方面显著高于GH基因型个体(P<0.05)。2. PPARδ基因多态性及其与秦川牛胴体、肉质性状的相关分析本研究对PPARδ基因的7个外显子进行SNP检测,结果仅发现了1个SNP位点,该突变发生在第2内含子,为61 C>T突变。在检测的6个群体中,均表现为KK基因型占主导地位,等位基因K为优势等位基因。南阳牛和夏南牛处于Hardy-Weinberg平衡状态( P>0.05 ),秦川牛、安格斯、鲁西牛和郏县红牛群体均处于Hardy-Weinberg不平衡状态( P<0.05 )。夏南牛群体遗传多态性较丰富,为中度多态,其余5个群体均属于低度多态。PPARδ基因61 C>T多态位点与秦川牛宰前活重和胴体重显著相关,表现为KK基因型个体宰前活重显著高于KL和LL基因型个体( P<0.05 ),KK基因型个体胴体长显著高于KL基因型个体。3. PPARγ基因多态性及其与秦川牛胴体、肉质性状的相关分析利用PCR-SSCP和DNA测序法对PPARγ基因的7个外显子及部分5’调控区进行了SNP筛查。4个新的SNP位点被发现,分别为外显子1的200 A>G,内含子2的59 C>G,外显子5的62 C>T,外显子7的74 G>T突变。PPARγ基因A>G多态位点位于第一外显子的200bp处,该位点导致天冬氨酸(D)转变为甘氨酸(G)。在南阳牛、秦川牛、郏县红牛、安格斯、鲁西牛和夏南牛6个群体中,等位基因A/B的频率分别为0.81/0.19、0.86/0.14、0.80/0.20、0.76/0.24、0.72/0.28和0.87/0.13。安格斯群体处于Hardy-Weinberg非平衡状态(P<0.05),其余5个群体均处于Hardy-Weinberg平衡状态(P>0.05)。秦川牛和夏南牛属于低度多态,南阳牛、郏县红牛、安格斯和鲁西牛属于中度多态。200 A>G位点与背膘厚及背部皮下脂肪厚显著相关,表现为AA基因型个体背膘厚和背部皮下脂肪厚显著高于AB基因型个体(P<0.05)。生物信息学研究发现,天冬氨酸转变为甘氨酸后,导致蛋白的二级结构在该处出现一个α螺旋的丢失,并且无规则卷曲变大。碱基突变导致该基因氨基酸疏水性增大,松散度减小。这些变化可能与该基因功能的变化有关。59C>G突变位于PPARγ基因第2内含子上,是在检测PPARγ基因第2外显子的突变时候发现的。该位点Y等位基因占主导地位,南阳牛、秦川牛、郏县红牛、安格斯、鲁西牛和夏南牛6个群体均处于Hardy-Weinberg不平衡状态(P<0.05)。南阳牛、郏县红牛、安格斯、鲁西牛和夏南牛5个群体属于中度多态,遗传多态性较丰富,秦川牛遗传多态性较低,为低度多态。并且研究发现该位点与秦川牛的背部皮下脂肪厚之间存在显著相关性。62C>T多态位点发生在PPARγ基因第5外显子上,6个群体均表现为II基因型的缺失,在南阳牛、秦川牛、郏县红牛、安格斯、鲁西牛和夏南牛6个群体中,等位基因J/I的频率分别为0.94/0.06、0.87/0.13、0.90/0.10、0.90/0.10、0.91/0.09和0.94/0.04。其中仅秦川牛处于Hardy-Weinberg不平衡状态(P<0.05),6个群体均表现为低度多态。相关性分析结果显示:JJ基因型的个体的背部皮下脂肪厚指标显著高于JI基因型个体(P<0.05)。74G>T位点导致了编码氨基酸由谷氨酰胺(Q)突变为组氨酸(H),生物信息学研究发现,氨基酸的突变导致该处一个β转角的丢失,无规则卷曲变小。疏水性增大,松散度细微减少。南阳牛、郏县红牛、秦川牛和鲁西牛群体处于Hardy-Weinberg平衡状态(P>0.05) ,安格斯和夏南牛群体处于Hardy-Weinberg不平衡状态(P<0.05)。南阳牛和夏南牛属于低度多态,其余4个群体属于中度多态。相关性分析结果显示:OP基因型个体胴体长显著高于OO和PP基因型个体(P<0.05),OO基因型个体背膘厚指标显著高于OP基因型个体(P<0.05),极显著高于PP基因型个体(P<0.01),OO基因型个体系水力显著低于OP和PP基因型个体(P<0.05)。4. PLIN基因多态性及其与秦川牛胴体、肉质性状的相关分析在牛PLIN基因的4个基因座首次检测到2处突变,分别为外显子3的156 T>C突变,外显子4的14 C>T突变。这2个位点在秦川牛群体中均处于中度多态,在156 T>C位点,AB基因型个体的宰前活体质量、胴体质量、背膘厚和眼肌面积均高于AA和BB基因型个体(P<0.05),而且AB基因型个体与AA基因型个体相比,在宰前活体质量、胴体质量、背膘厚和眼肌面积这4个指标方面达到差异极显著水平(P<0.01)。14 C>T突变相关性分析结果显示:DE基因型个体的胴体长、胴体胸深以及背膘厚显著高于DD和EE基因型个体(P<0.05),同时DD基因型个体肌肉嫩度极显著低于EE和DE基因型个体(P<0.01)。更多还原

【Abstract】 Genetic variations of lipid metabolism related genes (PPARα, PPARδ, PPARγ, and PLIN) were detected by PCR-SSCP, DNA sequencing and DNA sequence analysis in 717 individuals of six populations (Nanyang, Qinchuan, Jiaxian Red cattle, Luxi, Angus, and Xianan), and association analysis were carried out to evaluate the effects of genotypes of candidate genes on carcass and meat quality traits of 108 Qinchuan cattle. Molecular markers function was analyzed by bioinformation in missense mutation loci. The object of this study were to discover 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 PPARαgene and carcass, meat quality traits in Qinchuan cattleThe variations of eight loci in PPARαgene, including seven exons and intron2, were detected in six populations using PCR-SSCP and DNA sequencing. Six SNPs were detected for the first time.Three SNPs (2666 C>G, 2678 T>C, 2706 T>C) were detected, which located at the intron2 of PPARαgene. Allele gene D was predominant in the locus. The allele frequencies of D in Qinchuan, Nanyang, Jiaxian Red cattle, Luxi, Xianan and Augus populations were 0.571, 0.404, 0.419, 0.417, 0.422 and 0.386. And in the six populations, the SNP was moderate polymorphic, the PIC values were 0.421, 0.416, 0.434, 0.447, 0.438 and 0.413, respectively. The 2666 C>G locus was high significantly with loin muscle area (P<0.01), individuals with genotype CC had higher loin muscle area than those with genotype CD (P<0.01).T>C mutation was detected in the exon5 of PPARαgene by PCR-SSCP, which located at the juncture of exon5 and intron5, and the locus was synonymous mutation. The Chi-Square tests showed Nanyang, Jiaxian Red cattle, Augus, and Luxi cattle populations were at Hardy-Weinberg equilibrium (P>0.05), but Qinchuan cattle and Xianan cattle populations were at Hardy-Weinberg disequilibrium (P<0.05). The locus was moderate polymorphic in six populations, therefore the genetic polymorphisms from higher to lower were Qinchuan cattle, Jiaxian Red cattle, Augus, Luxi, Xianan and nanyang cattle. The 138T>C SNP had significant effects on loci muscle area, back fat thickness and hold water capability in Qinchuan cattle population.184 C>T mutation located at the seventh exon of PPARαgene. In this locus, Nanyang, Jiaxian Red cattle, Xianan, and Luxi cattle populations were at Hardy-Weinberg equilibrium (P>0.05), but Qinchuan cattle and Augus cattle populations were at Hardy-Weinberg disequilibrium (P<0.05). The index of genetics showed that Qinchuan cattle, Jiaxian Red cattle, Augus, Luxi and Xianan cattle populations belonged to low polymorphic and Nanyang cattle population was moderate polymorphic. The 184 C>T SNP was significantly associated with back fat thickness and carcass length (P<0.05).260 C>T mutation was detected in the eighth exon of PPARαgene. The allele frequencies of G/H were 0.925/0.075, 0.864/0.136, 0.791/0.209, 0.828/0.172, 0.835/0.165 and 0.853/0.147 in Qinchuan, Nanyang, Jiaxian Red cattle, Luxi, Xianan and Augus populations. Genotype II was no detected in Nanyang cattle population. And genotype GG was predominant, the frequencies of allele G was the highest in this locus. Nanyang cattle population was at Hardy-Weinberg equilibrium (P>0.05), but Qinchuan cattle, Jiaxian Red cattle, Augus, Luxi and Xianan cattle populations were at Hardy-Weinberg disequilibrium (P<0.05). Except for Jiaxian Red cattle (0.25<PIC=0.276<0.50), Qinchuan, Nanyang, Luxi, Xianan and Augus populations belonged to low polymorphic. The 260 C>T SNP was significantly associated with hold water capability (P<0.05). Individuals with genotype GG had higher hold water capability than those with genotype GH (P<0.05).2. Relationship between genetic variation of PPARδgene and carcass, meat quality traits in Qinchuan cattleThe seventh exons were detected, and only one SNP (61 C>T) was found in the intron2 in the PPARδgene. Genotype KK and allele K were predominant in the six populations. Nanyang and Xianan cattle populations were at Hardy-Weinberg equilibrium (P>0.05), but Qinchuan cattle, Jiaxian Red cattle, Augus and Luxi cattle populations were at Hardy-Weinberg disequilibrium (P<0.05). The 61 C>T SNP was moderate polymorphic in Xianan population, was low polymorphic in Qinchuan, Nanyang, Jiaxian Red cattle, Luxi and Augus populations. The 61 C>T SNP was significantly associated with Slaughter weight and Carcass weight (P<0.05) in Qinchuan cattle. Animals with genotype KK had higher Slaughter weight than those with genotypes KL and LL (P<0.05), individuals with genotype KK had higher carcass weight than those with genotype KL (P<0.05).3. Relationship between genetic variation of PPARγgene and carcass, meat quality traits in Qinchuan cattleFour SNPs (200 A>G (exon1), 59 C>G (intron2), 62 C>T (exon5) and 74 G>T (exon7)) of PPARγgene were detected in 8 loci, which located coding region and the part of 5’control region by PCR-SSCP and DNA sequencing.A>G SNP was detected, which located at 200 bp in the first exon of PPARγgene and resulted in D7G change. The allele frequencies of A/B were 0.81/0.19, 0.86/0.14, 0.80/0.20, 0.76/0.24, 0.72/0.28 and 0.87/0.13 in Qinchuan, Nanyang, Jiaxian Red cattle, Luxi, Xianan and Augus populations. In the six populations, Augus population was at Hardy-Weinberg disequilibrium (P<0.05). In Qinchuan cattle and Xianan cattle populations, the SNP was low polymorphic, but in Nanyang, Jiaxian Red cattle, Augus and Luxi cattle populaions, the SNP was moderate polymorphic. The 200 A>G SNP was significantly associated with back fat thickness and back subcutaneous fat thickness. Individuals with genotype AA had high back fat thickness and back subcutaneous fat thickness than those with genotype AB (P<0.05). Bioinformatics analysis showed that the mutation led Asp change into Gly, which had theα-helix lost, the coil wax, the hydrophobicity increase and loose degree decrease. This change may be related to the change of gene function.The 59 C>G SNP produced at intron2 of PPARγgene, which was found when detected the SNP of exon2. Allele Y was dominant in the six populations, all populations were at Hardy-Weinberg disequilibrium (P<0.05). In Nanyang, Jiaxian Red cattle, Augus, Luxi and Xianan cattle populations, the SNP was moderate polymorphic, but in Qinchuan cattle population, the SNP was low polymorphic. The 59 C>G SNP was had significant effects on back subcutaneous fat thickness.62 C>T mutation located at exon5 of PPARγgene, six populations all displayed the lost of genotype II. The allele frequencies of J/I were 0.94/0.06, 0.87/0.13, 0.90/0.10, 0.90/0.10, 0.91/0.09 and 0.94/0.04 in Qinchuan, Nanyang, Jiaxian Red cattle, Luxi, Xianan and Augus populations. Only Qinchuan cattle was at Hardy-Weinberg disequilibrium (P<0.05). All populations were at low polymorphic. Correlation analysis showed individuals with genotype JJ had higher back subcutaneous fat thickness than those with genotype JI (P<0.05).The 74 G>T mutation resulted Glu change into His. Bioinformatics analysis revealed there had theβ-sheet lost and coil decreased in 2D structure, and the hydrophobicity increased and loose degree decreased in the locus. Qinchuan cattle, Jiaxian Red cattle, Augus, Luxi and Nanyang cattle populations were at Hardy-Weinberg equilibrium (P>0.05), but Angus and Xianan cattle populations were at Hardy-Weinberg disequilibrium (P<0.05). In Nanyang cattle and Xianan cattle populations, the SNP was low polymorphic, in other populations, the SNP was moderate polymorphic. Animal with genotype OP was higher carcass length than those with genotypes OO and PP (P<0.05), individuals with genotype OO was higher mean values than these with genotypes OP (P<0.05) and PP (P<0.01) for back fat thickness, and animals with genotype OO was lower mean values than these with genotypes OP and PP (P<0.05) for hold water capability.4. Relationship between genetic variation of PLIN gene and carcass, meat quality traits in Qinchuan cattleA total of two SNPs (156 T>C (exon3), 14 C>T (exon4)) of PLIN gene were detected in 4 loci in Qinchuan cattle population. The two SNPs were belonging to moderate polymorphic. In the 156 T>C locus, animal with genotype AB was higher slaughter weight, carcass weight, back fat thickness and loin muscle area than those with genotypes AA and BB, and compared to individual with AA genotype was very significantly different(P<0.01). In the 14 C>T locus, this analysis also showed that the carcass length, carcass chest depth and back fat thickness of individual with DE genotype was significant higher than those with EE and DD genotypes (P<0.05), and compared to individual with EE and DE genotypes, the tenderness of individual with DD genotype was very significantly different(P<0.01).更多还原