肉牛RXRG和MyoD1基因的克隆、SNPs筛查及其与肉质和双胎性状的关联分析
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摘要
本研究分别以秦川牛、鲁西牛、西门塔尔牛和安格斯牛4个品种牛的514份血样和西门塔尔牛的组织样为材料,分别提取基因组DNA和RNA。运用生物信息学方法和同源序列克隆技术结合电子PCR (ePCR)、反转录PCR (RT-PCR)和限制性片段长度多态性PCR (PCR-RFLP),对牛的视黄素受体γ(RXRG)基因的cDNA序列和基因组DNA序列进行了克隆、鉴定与序列分析。运用测序和PCR-RFLP结合的方法对视黄素受体γ(RXRG)基因和生肌决定因子1 (MyoD1)基因的部分基因组DNA片段进行了单核苷酸多态检测,运用一般线性模型研究了MyoD1基因与上述6个品种牛部分生长与肉质性状的相关性,运用Logistic概率型非线性回归分析和卡方独立性检验研究了RXRG基因与上述6个品种牛双胎性状的相关性,得到了如下结果:
(1)利用RT-PCR技术,对牛RXRG基因进行克隆,将得到的片段重组到PMD19-T载体进行序列测定。获得了一个长为1811 bp的cDNA片断。通过氨基酸序列分析发现,牛RXRG基因的该片段由10个外显子组成,编码463个氨基酸。与其他动物同源性比较表明,该基因与人、猪、猕猴、小鼠、大鼠在氨基酸序列上分别有89.5%、93.4%、87.2%、83.9%、85.2%的同源性。获得了牛RXRG基因的cDNA序列。组织表达谱分析表明,牛RXRG基因在肌肉中高度表达,在其他组织中不表达。
(2)运用测序法寻找牛RXRG基因SNPs,筛查到一个新的多态位点A1941G,该位点位于3′非编码区。运用PCR-RFLP法验证并分析该位点在鲁西牛双胎群体和单胎群体及西门塔尔牛、安格斯牛和西蒙杂交牛单胎群体间的多态性,结果表明,在鲁西牛双胎和单胎群体中分布A、B两个等位基因,处于中度多态。经卡方适合性检验,鲁西双胎牛群体在该位点未达到Hardy-Weinberg平衡状态(P<0.05)。将鲁西牛群体的A1941G位点的基因型效应与双胎性状进行关联分析,卡方独立性检测结果显示,基因型分布在鲁西单、双胎牛群体上差异达到极显著水平(P<0.01)。
(3)运用测序法寻找牛MyoD1基因SNPs,筛查到一个新的多态位点C1867T,该位点C-T突变引起甘氨酸/丝氨酸的转变。运用PCR-RFLP法验证并分析该位点在不同群体间的多态性,并与相应牛群体的胴体性状进行关联分析。结果显示,西门塔尔牛、鲁西牛群体的C1867T位点的突变达到了Hardy-Weinberg平衡状态,而秦川牛未达到平衡状态。不同基因型对肉牛的背膘厚影响差异极显著(P<0.01),对大腿肉厚的影响显著(P<0.05),对日增重、屠宰率、肌间脂肪、大理石花纹、嫩度的影响差异不显著。
514 blood samples from 4 cattle populations-Qinchuan, Luxi, Simmental, and Angus were collected to extract genomic DNA, and some tissues samples from Simmental were taken to extract total RNA in this study. Then the isolation, identification and biologic information sequence analysis to the bovine RXRG gene and MyoD1 gene for the cDNA sequence and genomic sequence was performed by homology cloning and bioinformatics approach combined with electronic-PCR technique, RT-PCR and PCR-RFLP. And then, SNPs within some genomic fragments of the two gene were detected by sequencing. Finally, the association between RXRG and MyoD1 gene with some bovine twinning trait and meat quality traits in 6 populations were investigated. The main results were as follows:
1. Bovine RXRG gene cDNA by RT-PCR, the product was cloned in PMD19-T vector. The sequencing result showed that the fragment sequence coincided with the prediction, which is 1811 bp long. The analysis of amino acid sequence indicated that cattle RXRG gene consisted 10 exons and coded 463 amino acids. Homologous comparison with some animals indicated that cattle RXRG cDNA shared 89.5%, 93.4%, 87.2%, 83.9%, 85.2% similarity in nucleic acid sequence with human, pig, macaca, mouse and rattus. The cDNA of RXRG gene was cloned. RXRG cDNA was found in muscle tissue by the analysis of its expression in various tissues, but didn’t be found in other tissues.
2. A new SNP of cattle RXRG gene A1941G was detected by sequencing at 3′UTR. Different genotypes were determined in Luxi monotocous cows, Luxi twinning cows, Simmental cows, Angus cows and Simmental×Mongolia cows by restriction fragment length polymorphism (PCR-RFLP). The value of polymorphism information content indicated that this was a moderate polymorphism in Luxi monotocous cows and Luxi twinning cows. Theχ2 test indicated that the polymorphic locus in Luxi twinning cows did not fit Hardy-Weinberg equilibrium (P<0.05). Theχ2 test of associational analysis between genotypic distribution and twinning or monotocous trait in Luxi cows showed that the difference was very significant (P<0.01).
3. Based on gene pooling and sequencing, a new SNPs was found on the bovine MyoD1 gene, its transition C-T leads to a reaplacement of glicine with serine. Polymorphism and relationship with the carcass traits was analysised by PCR-RFLP in different populations. The transition of this locus fit in with Hardy-Weinberg equilibrium in Simmental cattle and Luxi cattle, but not in Qinchuan cattle. Statistical analysis revealed higher value in back fat and thickness of thigh (P<0.05), not in average daily growth, yield of carcass, intramuscular fat, marbling and yenderness.
(1)利用RT-PCR技术,对牛RXRG基因进行克隆,将得到的片段重组到PMD19-T载体进行序列测定。获得了一个长为1811 bp的cDNA片断。通过氨基酸序列分析发现,牛RXRG基因的该片段由10个外显子组成,编码463个氨基酸。与其他动物同源性比较表明,该基因与人、猪、猕猴、小鼠、大鼠在氨基酸序列上分别有89.5%、93.4%、87.2%、83.9%、85.2%的同源性。获得了牛RXRG基因的cDNA序列。组织表达谱分析表明,牛RXRG基因在肌肉中高度表达,在其他组织中不表达。
(2)运用测序法寻找牛RXRG基因SNPs,筛查到一个新的多态位点A1941G,该位点位于3′非编码区。运用PCR-RFLP法验证并分析该位点在鲁西牛双胎群体和单胎群体及西门塔尔牛、安格斯牛和西蒙杂交牛单胎群体间的多态性,结果表明,在鲁西牛双胎和单胎群体中分布A、B两个等位基因,处于中度多态。经卡方适合性检验,鲁西双胎牛群体在该位点未达到Hardy-Weinberg平衡状态(P<0.05)。将鲁西牛群体的A1941G位点的基因型效应与双胎性状进行关联分析,卡方独立性检测结果显示,基因型分布在鲁西单、双胎牛群体上差异达到极显著水平(P<0.01)。
(3)运用测序法寻找牛MyoD1基因SNPs,筛查到一个新的多态位点C1867T,该位点C-T突变引起甘氨酸/丝氨酸的转变。运用PCR-RFLP法验证并分析该位点在不同群体间的多态性,并与相应牛群体的胴体性状进行关联分析。结果显示,西门塔尔牛、鲁西牛群体的C1867T位点的突变达到了Hardy-Weinberg平衡状态,而秦川牛未达到平衡状态。不同基因型对肉牛的背膘厚影响差异极显著(P<0.01),对大腿肉厚的影响显著(P<0.05),对日增重、屠宰率、肌间脂肪、大理石花纹、嫩度的影响差异不显著。
514 blood samples from 4 cattle populations-Qinchuan, Luxi, Simmental, and Angus were collected to extract genomic DNA, and some tissues samples from Simmental were taken to extract total RNA in this study. Then the isolation, identification and biologic information sequence analysis to the bovine RXRG gene and MyoD1 gene for the cDNA sequence and genomic sequence was performed by homology cloning and bioinformatics approach combined with electronic-PCR technique, RT-PCR and PCR-RFLP. And then, SNPs within some genomic fragments of the two gene were detected by sequencing. Finally, the association between RXRG and MyoD1 gene with some bovine twinning trait and meat quality traits in 6 populations were investigated. The main results were as follows:
1. Bovine RXRG gene cDNA by RT-PCR, the product was cloned in PMD19-T vector. The sequencing result showed that the fragment sequence coincided with the prediction, which is 1811 bp long. The analysis of amino acid sequence indicated that cattle RXRG gene consisted 10 exons and coded 463 amino acids. Homologous comparison with some animals indicated that cattle RXRG cDNA shared 89.5%, 93.4%, 87.2%, 83.9%, 85.2% similarity in nucleic acid sequence with human, pig, macaca, mouse and rattus. The cDNA of RXRG gene was cloned. RXRG cDNA was found in muscle tissue by the analysis of its expression in various tissues, but didn’t be found in other tissues.
2. A new SNP of cattle RXRG gene A1941G was detected by sequencing at 3′UTR. Different genotypes were determined in Luxi monotocous cows, Luxi twinning cows, Simmental cows, Angus cows and Simmental×Mongolia cows by restriction fragment length polymorphism (PCR-RFLP). The value of polymorphism information content indicated that this was a moderate polymorphism in Luxi monotocous cows and Luxi twinning cows. Theχ2 test indicated that the polymorphic locus in Luxi twinning cows did not fit Hardy-Weinberg equilibrium (P<0.05). Theχ2 test of associational analysis between genotypic distribution and twinning or monotocous trait in Luxi cows showed that the difference was very significant (P<0.01).
3. Based on gene pooling and sequencing, a new SNPs was found on the bovine MyoD1 gene, its transition C-T leads to a reaplacement of glicine with serine. Polymorphism and relationship with the carcass traits was analysised by PCR-RFLP in different populations. The transition of this locus fit in with Hardy-Weinberg equilibrium in Simmental cattle and Luxi cattle, but not in Qinchuan cattle. Statistical analysis revealed higher value in back fat and thickness of thigh (P<0.05), not in average daily growth, yield of carcass, intramuscular fat, marbling and yenderness.
引文
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