貉子MC4R、POU1F1基因多态性及其与生长性状关系的研究
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摘要
貉子是非常珍贵的毛皮动物,貉皮柔软轻便、美观大方,具有很高的经济价值。貉的副产品也有很高的开发利用价值。目前,我国养貉业发展迅速,貉产品在国际市场上占有重要地位。国内外对貉子养殖技术已经做了大量研究,饲料、管理和疾病防治等技术已经相当成熟,但是貉的育种还一直沿用传统方法,不但周期长、费用高而且不精确。20世纪80年代开始,DNA多态性等各种遗传标记方法的迅速发展,使得分子遗传标记应用于动物育种成为现实。
动物的生长性状是受多基因控制的,经过大量的试验证明MC4R和POUIF1基因对动物的生长具有重要的调节作用。MC4R基因是调节能量平衡与能量动态平衡的重要信号分子,能够控制动物的食欲、体重以及能量代谢POUIFI作为反式因子通过激活生长激素基因(GH)、催乳素基因(PRL)和促甲状腺素基因亚基(TSHβ)的转录,对生物的生长发育产生重要影响。因此,本试验选取MC4R和POUIFI基因为候选基因,以周家养殖场的226只貉子为实验对象,用PCR-SSCP法,检测整个MC4R和部分POUIFI基因的SNPs,并分析各突变位点导致的多态性与生长性状之间的相关性。主要研究结果如下：
1.MC4R基因的研究结果
(1)克隆了整个MC4R基因共1239bp,将此基因序列通过Genbank中的BLAST比较分析表明与人的MC4R基因同源性是88%；与狐狸和犬MC4R基因同源性均是98%；与熊猫MC4R基因的同源性是92%；与野猪MC4R基因的同源性是87%。
(2)在MC4R-1引物内检测出三个突变位点A43G、A143G和T241C,三个突变位点产生AA、AB和BB三种基因型。经多重比较表明,BB型个体体重和胸围高于AA型和AB型,且差异显著(P<0.05)；AA型和AB型之间差异不显著(P>0.05)。
(3)MC4R-6引物的PCR产物经SSCP分析,检测到CC、CD和DD三种基因型,经克隆测序分析发现是由A118C突变引起的。经最小二乘法分析表明,此突变对整个试验群体体重有影响(P<0.2)；在雄性个体中,对体长有显著影响(P<0.05)；对其余生长性状均无影响(P>0.2)。
(4)MC4R-7引物的PCR产物经SSCP分析,检测到EE、EF和FF三种基因型,经克隆测序分析发现是由A42G和C114A突变引起的。最小二乘分析结果表明,这两个突变位点对整个试验群体体重和体长都有影响(P<0.2)；对其它各生长性状均无影响(P>0.2)。
(5)合并基因型后统计分析发现：BBEF为最优基因型,平均体重为6.224kg,要明显高于BB型(平均体重5.899kg)和EF型(平均体重5.320kg)。ABFF型的貉子平均体重最低为5.000kg。
2.POU1F1基因的研究结果
(1)克隆了貉子POU1F1基因第4外显子,序列长度别为150bp,已将该序列提交到GenBank上(Accession No:GU361626)。将所测序列通过GeneBank中的Blast比较分析,此段序列与犬的同源性为100%；与牛的同源性分别为94%；与人和黑猩猩的同源性是92%；与褐鼠的同源性是88%；与斑马鱼同源性为84%；与原鸡的同源性为83%。
(2)用SSCP方法对此片段进行多态性检测,在不同试验个体中均未检测出多态。
Raccoon dog whose fur is soft, light, beautiful and generous is a very valuable fur animal with high economic value. Its by-product has high development and utilization of value. Currently, the breeding industry of raccoon develops rapidly and raccoon product occupies an important position in the international market. There are numerous studies about breeding industry and feed, management and disease prevention and control technology has been quite mature. But breeding of raccoon has also been the traditional method, that method has not only a long cycle, but also has high cost and inaccuracy. From the 1980s, the rapid development of various genetic markers as DNA polymorphism makes molecular genetic markers used in animal breeding.
Growth traits of animals is controlled by several genes, it is proved that MC4R and POU1F1 gene has an important regulatory role on the growth of animals after a number of tests. MC4R gene which is an important signaling molecule of regulation of energy balance and energy homeostasis can control animal appetite, weight and energy metabolism. As a trans-factor POU1F1 has important effects on growth and development of life by activating transcription of growth hormone gene (GH), prolactin gene (PRL) and thyrotrophic gene subunit (TSH(3). Therefore, this test selects MC4R and POU1F1 genes as a candidate gene, and 226 raccoons of Zhoujia farm are as experimental subjects, SNPs of the whole MC4R and part of POU1F1 gene is detected by PCR-SSCP method, and the correlation between polymorphism and growth traits caused by each mutation is analyzed. The main results are as follows:
1. MC4R gene research results
(1) The whole MC4R genes 1239bp are cloned, the homology of MC4R gene with human is 88% by BLAST comparative analysis in Genbank; the homology of MC4R gene with fox and dog is 98%; the homology of MC4R gene with panda is 92%; the homology of MC4R gene with wild boar is 87%.
(2) Three mutations A43G, A143G and T241C are detected in MC4R-1 primer, the three mutation sites generate AA, AB and BB genotypes. The multiple comparisons show that:weight and bust of BB-type are higher than AA-type and AB-type, the difference is significant (P<0.05); the difference between AA-type and AB-type is not significant (P>0.05).
(3) CC, CD and DD genotypes are detected by SSCP analysis product of MC4R-6 PCR of primers, it causes by A118C mutant by clone and sequence analysis. The least squares analysis shows that:the mutations affect weight of the whole test group (P<0.2); in males, it has a significant effect on the length of the body (P <0.05) and has no effect on the rest of growth traits (P>0.2).
(4) EE, EF and FF genotypes are detected by SSCP analysis product of MC4R-7 PCR of primers, it causes by A42G and Cl 14A mutant by clone and sequence analysis. The least squares analysis shows that:the two mutations affect weight and body length of the whole test group (P<0.2) and has no effect on the rest of growth traits (P>0.2).
(5) Statistical analysis after combining genotypes shows that:BBEF is the optimal genotype, the average weight is 6.224kg, it is significantly higher than BB-type (the average weight is 5.899kg) and EF-type (the average weight is 5.320kg). The minimum weight of ABFF-type raccoon is 5.000kg.
2. POU1F1 gene research results
(1) Exon 4 of raccoon POU1F1 gene is cloned, the sequence length is 150bp, the sequence has been submitted to GenBank (Accession No:GU361626). The homology of this section with dog is 100% by BLAST comparative analysis in Genbank; the homology of this section with cattle is 94%; the homology of this section with chimpanzee and human is 92%; the homology of this section with brown rat is 88%; the homology of this section with zebra fish is 84%; the homology of this section with jungle fowl is 83%.
(2) Polymorphism detection is done for this section with SSCP method, there is no polymorphism detected in different test individuals.
动物的生长性状是受多基因控制的,经过大量的试验证明MC4R和POUIF1基因对动物的生长具有重要的调节作用。MC4R基因是调节能量平衡与能量动态平衡的重要信号分子,能够控制动物的食欲、体重以及能量代谢POUIFI作为反式因子通过激活生长激素基因(GH)、催乳素基因(PRL)和促甲状腺素基因亚基(TSHβ)的转录,对生物的生长发育产生重要影响。因此,本试验选取MC4R和POUIFI基因为候选基因,以周家养殖场的226只貉子为实验对象,用PCR-SSCP法,检测整个MC4R和部分POUIFI基因的SNPs,并分析各突变位点导致的多态性与生长性状之间的相关性。主要研究结果如下：
1.MC4R基因的研究结果
(1)克隆了整个MC4R基因共1239bp,将此基因序列通过Genbank中的BLAST比较分析表明与人的MC4R基因同源性是88%；与狐狸和犬MC4R基因同源性均是98%；与熊猫MC4R基因的同源性是92%；与野猪MC4R基因的同源性是87%。
(2)在MC4R-1引物内检测出三个突变位点A43G、A143G和T241C,三个突变位点产生AA、AB和BB三种基因型。经多重比较表明,BB型个体体重和胸围高于AA型和AB型,且差异显著(P<0.05)；AA型和AB型之间差异不显著(P>0.05)。
(3)MC4R-6引物的PCR产物经SSCP分析,检测到CC、CD和DD三种基因型,经克隆测序分析发现是由A118C突变引起的。经最小二乘法分析表明,此突变对整个试验群体体重有影响(P<0.2)；在雄性个体中,对体长有显著影响(P<0.05)；对其余生长性状均无影响(P>0.2)。
(4)MC4R-7引物的PCR产物经SSCP分析,检测到EE、EF和FF三种基因型,经克隆测序分析发现是由A42G和C114A突变引起的。最小二乘分析结果表明,这两个突变位点对整个试验群体体重和体长都有影响(P<0.2)；对其它各生长性状均无影响(P>0.2)。
(5)合并基因型后统计分析发现：BBEF为最优基因型,平均体重为6.224kg,要明显高于BB型(平均体重5.899kg)和EF型(平均体重5.320kg)。ABFF型的貉子平均体重最低为5.000kg。
2.POU1F1基因的研究结果
(1)克隆了貉子POU1F1基因第4外显子,序列长度别为150bp,已将该序列提交到GenBank上(Accession No:GU361626)。将所测序列通过GeneBank中的Blast比较分析,此段序列与犬的同源性为100%；与牛的同源性分别为94%；与人和黑猩猩的同源性是92%；与褐鼠的同源性是88%；与斑马鱼同源性为84%；与原鸡的同源性为83%。
(2)用SSCP方法对此片段进行多态性检测,在不同试验个体中均未检测出多态。
Raccoon dog whose fur is soft, light, beautiful and generous is a very valuable fur animal with high economic value. Its by-product has high development and utilization of value. Currently, the breeding industry of raccoon develops rapidly and raccoon product occupies an important position in the international market. There are numerous studies about breeding industry and feed, management and disease prevention and control technology has been quite mature. But breeding of raccoon has also been the traditional method, that method has not only a long cycle, but also has high cost and inaccuracy. From the 1980s, the rapid development of various genetic markers as DNA polymorphism makes molecular genetic markers used in animal breeding.
Growth traits of animals is controlled by several genes, it is proved that MC4R and POU1F1 gene has an important regulatory role on the growth of animals after a number of tests. MC4R gene which is an important signaling molecule of regulation of energy balance and energy homeostasis can control animal appetite, weight and energy metabolism. As a trans-factor POU1F1 has important effects on growth and development of life by activating transcription of growth hormone gene (GH), prolactin gene (PRL) and thyrotrophic gene subunit (TSH(3). Therefore, this test selects MC4R and POU1F1 genes as a candidate gene, and 226 raccoons of Zhoujia farm are as experimental subjects, SNPs of the whole MC4R and part of POU1F1 gene is detected by PCR-SSCP method, and the correlation between polymorphism and growth traits caused by each mutation is analyzed. The main results are as follows:
1. MC4R gene research results
(1) The whole MC4R genes 1239bp are cloned, the homology of MC4R gene with human is 88% by BLAST comparative analysis in Genbank; the homology of MC4R gene with fox and dog is 98%; the homology of MC4R gene with panda is 92%; the homology of MC4R gene with wild boar is 87%.
(2) Three mutations A43G, A143G and T241C are detected in MC4R-1 primer, the three mutation sites generate AA, AB and BB genotypes. The multiple comparisons show that:weight and bust of BB-type are higher than AA-type and AB-type, the difference is significant (P<0.05); the difference between AA-type and AB-type is not significant (P>0.05).
(3) CC, CD and DD genotypes are detected by SSCP analysis product of MC4R-6 PCR of primers, it causes by A118C mutant by clone and sequence analysis. The least squares analysis shows that:the mutations affect weight of the whole test group (P<0.2); in males, it has a significant effect on the length of the body (P <0.05) and has no effect on the rest of growth traits (P>0.2).
(4) EE, EF and FF genotypes are detected by SSCP analysis product of MC4R-7 PCR of primers, it causes by A42G and Cl 14A mutant by clone and sequence analysis. The least squares analysis shows that:the two mutations affect weight and body length of the whole test group (P<0.2) and has no effect on the rest of growth traits (P>0.2).
(5) Statistical analysis after combining genotypes shows that:BBEF is the optimal genotype, the average weight is 6.224kg, it is significantly higher than BB-type (the average weight is 5.899kg) and EF-type (the average weight is 5.320kg). The minimum weight of ABFF-type raccoon is 5.000kg.
2. POU1F1 gene research results
(1) Exon 4 of raccoon POU1F1 gene is cloned, the sequence length is 150bp, the sequence has been submitted to GenBank (Accession No:GU361626). The homology of this section with dog is 100% by BLAST comparative analysis in Genbank; the homology of this section with cattle is 94%; the homology of this section with chimpanzee and human is 92%; the homology of this section with brown rat is 88%; the homology of this section with zebra fish is 84%; the homology of this section with jungle fowl is 83%.
(2) Polymorphism detection is done for this section with SSCP method, there is no polymorphism detected in different test individuals.
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