藏鸡三个生长相关基因多态性研究暨Slc24a5基因与鸡黑色素沉积关系初步研究
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摘要
本论文由两个部分组成:藏鸡LEPR、IGF-2、MC4R基因SNP筛查及其与部分生长性状关联分析;Slc24a5基因与鸡黑色素沉积关系初步研究。
第一部分:藏鸡是我国以原始饲养状态保存下来的珍贵的高原地方鸡种。适应长期低压、缺氧、低温恶劣环境的藏鸡有着初生生长速度慢,成年个体小的显著特点。一方面,藏鸡同平原家禽一样,生长发育主要受脑垂体生长轴以及采食能量代谢平衡系统控制;同时,低氧环境也对动物的生长有很大影响。为了揭示藏鸡独特生长特点的遗传机制,本研究以脑垂体生长轴上的IGF-2基因、控制能量代谢的MC4R基因,以及既参与调节能量代谢又与低氧适应相关的LEPR基因作为控制藏鸡生长发育性状主基因的候选基因,利用DNA混合池直接测序寻找潜在单核苷酸多态性(SNP),并采用PCR-RFLP,Mp-PCR,M-ASP方法对SNPs在藏鸡和隐性白羽鸡群体中进行检测,分析这些基因的遗传多样性及突变位点在两群体中基因型与等位基因分布趋势。同时结合藏鸡群体生长发育性状,研究了3个基因不同基因型对藏鸡生长发育的可能影响。主要结果如下:
1.对包含鸡LEPR基因的leptin结合区(LBD)编码区在内的第7至14外显子进行了SNPs筛查,在该基因的第13外显子和14外显子上分别发现一个多态位点:A18697和A1921G。其中对于A1921G,3种基因型频率在2个品种间存在极显著差异(P＜0.01)。与藏鸡部分生长性状关联分析表明,A1896T对藏鸡的初生重、2周龄体重、7周龄体重、7周龄体斜长都有显著影响,A1921G对藏鸡的初生重、初胫围、2周龄体重、2周龄胫围、4周龄体重均有显著影响,其中含有G等位基因个体的初生生长性状值显著低于AA型个体.推测该位点氨基酸的改变(Thr641Ala)可能影响LEPR的LBD区与Leptin的结合。
2.对IGF-2基因整个编码区进行了SNP分析,在该基因第2外显子上发现一个同义突变A126T。与藏鸡部分生长性状关联分析表明,该位点对藏鸡的2周龄胫围有显著影响,AA型个体的第2周龄胫围要显著高于AG型与GG型个体。
3.对MC4R基因整个编码区进行了SNP分析,发现一个同义突变G315T,并且发现在隐性白羽鸡中没有T等位基因的存在,而在藏鸡中存在3种基因型的个体。与藏鸡部分生长性状关联分析结果表明,该位点对藏鸡的7周龄体斜长和16周龄胫围均有显著影响。GG型个体的7周龄体斜长和16周龄胫围均显著大于其他GT型和TT型个体。
第二部分:泰和乌骨鸡是中国优良的地方品种,除了有优质肉蛋品质外,乌骨鸡与普通鸡种最明显的差异是乌骨鸡体内含有大量黑色素,虽然国内外研究者对其体内黑色素的沉积分布规律进行了广泛的研究,但对于控制其黑色素沉积规律的具体遗传机制仍不清楚。最近,在斑马鱼和人中的研究表明,Slc24a5基因在控制肤色方面起重要作用,该基因可能通过其编码区的碱基的突变而造成斑马鱼野生型和突变型以及黑人与白人肤色的差异。为了验证鸡Slc24a5基因是否以类似的机制控制鸡肤色的变化,本研究以泰合乌骨鸡为材料,以体内黑色素沉积很少的尤溪麻鸡作为对照,利用测序寻找在两个鸡种间是否存在Slc24a5基因编码区碱基差异,并研究了Slc24a5基因在两鸡种多个组织的表达情况以分析该基因与黑色素沉积的可能关系。初步结果如下:
1.比较了两个鸡种6个个体(每品种3个)Slc24a5基因编码区的碱基序列。经分析,没有找到在两品种间特异的碱基差异。说明不同于人和斑马鱼中的研究,鸡肤色差异并不是由该基因编码区的碱基突变引起的。
2.在两鸡种中组织表达谱分析结果表明,Slc24a5基因在鸡组织中的表达模式与斑马鱼相似,即在黑色素沉积程度愈高的组织(器官)Slc24a5表达愈多(在泰和乌骨鸡的眼、皮肤、肌肉均大量表达;在尤溪麻鸡的眼大量表达,其它组织较少)。提示在斑马鱼和鸡中,Slc24a5基因在黑色素代谢途径中可能起着类似作用。
This thesis includes two parts:SNPs detection of LEPR,IGF-2,MC4R genes in Tibetan Chicken and association analysis with their Growth traits;A preliminary study of the relationship between Slc24a5 gene and chicken melanin pigmentation.
In partⅠ,LEPR,IGF-2,MC4R genes were used as the main effect candidate gene of growth traits of Tibet chicken.Single nucleotide polymorphisms(SNPs) were detected by DNA pooling and sequencing methods.Then we tested these SNPs in both Recessive White(RW) and Tibetan chicken(TC) by PCR-RFLP,Mp-RFLP or M-ASP techniques. And the Genotype frequencies and Alle frequencies were counted.Meanwhile,their association with partial growth traits of Tibetan chicken was analyzed.The results are as follows:
1.Two SNPs in LEPR gene were found:A1869T and A1921G.A1869T is a nonsense mutation in exon13,a base pair mutation from A to T changes PvuII identifying site. Three genotypes exist in both RW and TC,and there are significant associations between the genotype of A1869T with some growth traits of TC.(Body weight at birth, Body weight at 2 weeks of age,Body weight at 7 weeks of age and Body length at 7 weeks of age)(p＜0.05);A1921G is a missense mutation in exon14,a base pair mutation from A to G changes Thr to Ala.On this site,there are striking differencies of Genotype frequencies between two chicken breeds,and significant associations between the genotype of A1921G with some growth traits of TC.(Body weight at birth,Shank girth at birth,Body weight at 2 weeks of age,Shank girth at 2 weeks of age,Body weight at 4 weeks of age)(p＜0.05)
2.One SNP A126T in IGF-2 gene were found.A126T is a nonsense mutation in exon2, there are significant associations between the genotypes of A126T with Shank girth at 2 weeks of age of TC(p＜0.05).
3.MC4R polymorphism G315T have significant(P＜0.05) effect on Body length at 7 weeks of age and Shank girth at 16 weeks of age in TC.
In partⅡ,we analyzed the relationship between chicken Slc24a5 gene and chicken melanin pigmentation.
1.The whole coding region of chicken Slc24a5 gene were sequenced in two Chinese native chicken breeds different in skin color.And no possible causal mutation responsible for the skin color variation was identified.It seems that unlike its human or zebrafish orthologs,the skin color difference between Taihe Chicken and Youxi Chicken is not caused by coding region mutations in chicken Slc24a5.
2.We also found Slc24a5 is highly expressed in tissues highly melanin pigmented(like eyes,skin,muscle of Taihe Chicken and eyes of Youxi Chicken);and poorly expressed in tissues less melanin pigmented(like heart and brain of Taihe Chicken) or hardly melanin pigmented(like most Youxi Chicken tissues except eyes).Theses results suggest chicken Slc24a5,like its human and zebrafish orthologs,appears to be highly expressed in melanin-producing cells.
第一部分:藏鸡是我国以原始饲养状态保存下来的珍贵的高原地方鸡种。适应长期低压、缺氧、低温恶劣环境的藏鸡有着初生生长速度慢,成年个体小的显著特点。一方面,藏鸡同平原家禽一样,生长发育主要受脑垂体生长轴以及采食能量代谢平衡系统控制;同时,低氧环境也对动物的生长有很大影响。为了揭示藏鸡独特生长特点的遗传机制,本研究以脑垂体生长轴上的IGF-2基因、控制能量代谢的MC4R基因,以及既参与调节能量代谢又与低氧适应相关的LEPR基因作为控制藏鸡生长发育性状主基因的候选基因,利用DNA混合池直接测序寻找潜在单核苷酸多态性(SNP),并采用PCR-RFLP,Mp-PCR,M-ASP方法对SNPs在藏鸡和隐性白羽鸡群体中进行检测,分析这些基因的遗传多样性及突变位点在两群体中基因型与等位基因分布趋势。同时结合藏鸡群体生长发育性状,研究了3个基因不同基因型对藏鸡生长发育的可能影响。主要结果如下:
1.对包含鸡LEPR基因的leptin结合区(LBD)编码区在内的第7至14外显子进行了SNPs筛查,在该基因的第13外显子和14外显子上分别发现一个多态位点:A18697和A1921G。其中对于A1921G,3种基因型频率在2个品种间存在极显著差异(P＜0.01)。与藏鸡部分生长性状关联分析表明,A1896T对藏鸡的初生重、2周龄体重、7周龄体重、7周龄体斜长都有显著影响,A1921G对藏鸡的初生重、初胫围、2周龄体重、2周龄胫围、4周龄体重均有显著影响,其中含有G等位基因个体的初生生长性状值显著低于AA型个体.推测该位点氨基酸的改变(Thr641Ala)可能影响LEPR的LBD区与Leptin的结合。
2.对IGF-2基因整个编码区进行了SNP分析,在该基因第2外显子上发现一个同义突变A126T。与藏鸡部分生长性状关联分析表明,该位点对藏鸡的2周龄胫围有显著影响,AA型个体的第2周龄胫围要显著高于AG型与GG型个体。
3.对MC4R基因整个编码区进行了SNP分析,发现一个同义突变G315T,并且发现在隐性白羽鸡中没有T等位基因的存在,而在藏鸡中存在3种基因型的个体。与藏鸡部分生长性状关联分析结果表明,该位点对藏鸡的7周龄体斜长和16周龄胫围均有显著影响。GG型个体的7周龄体斜长和16周龄胫围均显著大于其他GT型和TT型个体。
第二部分:泰和乌骨鸡是中国优良的地方品种,除了有优质肉蛋品质外,乌骨鸡与普通鸡种最明显的差异是乌骨鸡体内含有大量黑色素,虽然国内外研究者对其体内黑色素的沉积分布规律进行了广泛的研究,但对于控制其黑色素沉积规律的具体遗传机制仍不清楚。最近,在斑马鱼和人中的研究表明,Slc24a5基因在控制肤色方面起重要作用,该基因可能通过其编码区的碱基的突变而造成斑马鱼野生型和突变型以及黑人与白人肤色的差异。为了验证鸡Slc24a5基因是否以类似的机制控制鸡肤色的变化,本研究以泰合乌骨鸡为材料,以体内黑色素沉积很少的尤溪麻鸡作为对照,利用测序寻找在两个鸡种间是否存在Slc24a5基因编码区碱基差异,并研究了Slc24a5基因在两鸡种多个组织的表达情况以分析该基因与黑色素沉积的可能关系。初步结果如下:
1.比较了两个鸡种6个个体(每品种3个)Slc24a5基因编码区的碱基序列。经分析,没有找到在两品种间特异的碱基差异。说明不同于人和斑马鱼中的研究,鸡肤色差异并不是由该基因编码区的碱基突变引起的。
2.在两鸡种中组织表达谱分析结果表明,Slc24a5基因在鸡组织中的表达模式与斑马鱼相似,即在黑色素沉积程度愈高的组织(器官)Slc24a5表达愈多(在泰和乌骨鸡的眼、皮肤、肌肉均大量表达;在尤溪麻鸡的眼大量表达,其它组织较少)。提示在斑马鱼和鸡中,Slc24a5基因在黑色素代谢途径中可能起着类似作用。
This thesis includes two parts:SNPs detection of LEPR,IGF-2,MC4R genes in Tibetan Chicken and association analysis with their Growth traits;A preliminary study of the relationship between Slc24a5 gene and chicken melanin pigmentation.
In partⅠ,LEPR,IGF-2,MC4R genes were used as the main effect candidate gene of growth traits of Tibet chicken.Single nucleotide polymorphisms(SNPs) were detected by DNA pooling and sequencing methods.Then we tested these SNPs in both Recessive White(RW) and Tibetan chicken(TC) by PCR-RFLP,Mp-RFLP or M-ASP techniques. And the Genotype frequencies and Alle frequencies were counted.Meanwhile,their association with partial growth traits of Tibetan chicken was analyzed.The results are as follows:
1.Two SNPs in LEPR gene were found:A1869T and A1921G.A1869T is a nonsense mutation in exon13,a base pair mutation from A to T changes PvuII identifying site. Three genotypes exist in both RW and TC,and there are significant associations between the genotype of A1869T with some growth traits of TC.(Body weight at birth, Body weight at 2 weeks of age,Body weight at 7 weeks of age and Body length at 7 weeks of age)(p＜0.05);A1921G is a missense mutation in exon14,a base pair mutation from A to G changes Thr to Ala.On this site,there are striking differencies of Genotype frequencies between two chicken breeds,and significant associations between the genotype of A1921G with some growth traits of TC.(Body weight at birth,Shank girth at birth,Body weight at 2 weeks of age,Shank girth at 2 weeks of age,Body weight at 4 weeks of age)(p＜0.05)
2.One SNP A126T in IGF-2 gene were found.A126T is a nonsense mutation in exon2, there are significant associations between the genotypes of A126T with Shank girth at 2 weeks of age of TC(p＜0.05).
3.MC4R polymorphism G315T have significant(P＜0.05) effect on Body length at 7 weeks of age and Shank girth at 16 weeks of age in TC.
In partⅡ,we analyzed the relationship between chicken Slc24a5 gene and chicken melanin pigmentation.
1.The whole coding region of chicken Slc24a5 gene were sequenced in two Chinese native chicken breeds different in skin color.And no possible causal mutation responsible for the skin color variation was identified.It seems that unlike its human or zebrafish orthologs,the skin color difference between Taihe Chicken and Youxi Chicken is not caused by coding region mutations in chicken Slc24a5.
2.We also found Slc24a5 is highly expressed in tissues highly melanin pigmented(like eyes,skin,muscle of Taihe Chicken and eyes of Youxi Chicken);and poorly expressed in tissues less melanin pigmented(like heart and brain of Taihe Chicken) or hardly melanin pigmented(like most Youxi Chicken tissues except eyes).Theses results suggest chicken Slc24a5,like its human and zebrafish orthologs,appears to be highly expressed in melanin-producing cells.
引文
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