鸡多趾候选基因的SNPs及表达差异研究
摘要
多趾存在于少数鸡种中,便于实验操作和观察,是研究肢体发育过程中基因之间相互作用的极好材料。参考人和鼠多趾相关基因间的相互作用以及多趾同源区间,我们将En2、Shh、Lmbr1、Hlxb9和Gli3基因作为鸡多趾研究的候选基因,寻找与多趾有关的SNPs,并检测候选基因表达上的差异,希望揭示候选基因间的相互作用并阐明鸡多趾形成的原因。
SNP搜寻所用实验材料共6组,有丝羽乌骨鸡(五趾)、A系肉鸡(四趾)、四趾菊花鸡、五趾菊花鸡、四趾北京油鸡和五趾北京油鸡。每组选5个个体对候选基因进行PCR后分6组混合回收产物并测序比对,在五个候选基因的外显子、内含子保守区以及Lmbr1的5’上游区寻找能严格区别四趾和五趾表型的SNPs作为趾型相关SNPs。结果显示这五个候选基因的外显子相当保守,只发现Lmbr1外显子16的一处C/T变异和Gli3外显子15的G/A变异,没有造成氨基酸改变,但测序比对或SSCP筛查排除了它们与趾型的关系。在候选基因的内含子保守区我们只发现Lmbr1的内含子5有一处C/A变异与趾型相关,定位在chr2:7901586bp。所有的四趾鸡该位点都为C,所有的五趾鸡该位点都为A/N。对菊花鸡、北京油鸡、CAU资源家系(由A系肉鸡和丝羽乌骨鸡建立)共517个体中进行了SSCP群体分型,证实了这个SNP的确与趾型相关。在Lmbr1 5’上游区段的筛查中,我们从26处变异中筛查出6处趾型相关SNP,并将这6处SNP定位在Chr2:7922655～Chr2:7928742bp之间。
在MatInspector网上预测中,Lmbr1 5’上游的6处SNP中有3处改变了同源异形蛋白因子或胚胎形成有关的蛋白结合位点。Lmbr1内含子5的变异至少改变了3个同源异形蛋白因子的结合位点,为其编码的Hox基因家族在人和鼠多趾的发育与模式形成中起着重要作用。预测结果也支持Lmbr1与趾型有关。
候选基因的表达研究采用白莱航(四趾)和丝羽乌骨鸡(五趾)的纯繁种蛋同批孵化,选择同期发育的胚胎分为头、躯体和肢芽三部分提取RNA。采用RT-PCR法,我们最早在1d(孵化24小时)检测到Gli3、Lmbr1和Shh的表达,早于胚胎原位杂
Polydactyly is a character in Several chicken breeds, which is a good material to study the interaction among genes in the process of limb growth for it is easy to operate and observate .Considering the interaction among polydactyly relevant genes and the polydactyly homologous region in human and mouse, we selected En2, Shh, Lmbr1 , Hlxb9 and Gli3 as polydactyly candidate genes in chicken. The SNPs related to toes and the difference in the genes expression were found and detected, in order to uncover the functions of candidate genes and explain the mechanism of chicken polydactyly..The materials used for finding SNPs included six groups:Silkies(5-toed), A strain of broilers (4-toed), Juhua chickens (4-teod and 5-teod) and Beijing-youji chickens (4-teod and 5-teod) .With 5 chickens from each group, exons and intron-conserved-domains (ICDs ) of five candidate genes, and 5' upstream of Lmbr1 were amplified separately. Then each sequence were purified and compared by 6 groups to search for the SNPs which could strictly distinguish 4-toed from 5-toed. The exons of the 5 genes were conservative and only two SNPs were found , one SNP (C/T) in exonl6 of Lmbr1 , the other SNP (G/A) in exon15 of Gli3. More sequences comparison and SSCP results exclude their relation to toes. But in ICDs, only one SNP(C/A) in Lmbrl intron5 was found and mapped to chr2:7901586bp, C variation correlating with 4-toed and A variation correlating with 5-toed, which was consistent with SSCP typing results from 517 DNAs of Juhua chickens, Beijing-youji chickens, CAU resource reference population(established by Silkies and A strain of broilers) .From the 5'-upstream of Lmbrl , 6 out of 26 SNPs were found related to toes, mapped to Chr2:7922655~ Chr2:7928742bp.Accordong to internet MatInspector prediction, 3 of the 6 SNPs could change homeodomain factor or protein binding sites that relevant to embryogenesis; the SNP in the intron5 of Lmbrl could change three homeodomain transcription factors binding sites. Their coding genes. Hox gene family, play important roles in the anterior-posterior axis in the development of polydactyly in human and mouse. The prediction results supported the influence of Lmbrl in the development of toes.To detect the differences in candidate genes' expression, RNAs were
used, which were extracted from heads, bodies and limb buds of White Leghon embryos (4-toed) and Silkie embryos (5-toed) at the same stage.The expression of GHS, Lmbrl and Shh was initially detected in Id (24h incubation) by RT-PCR, ahead of the first detectable time 2.5d~3d in Whole-mount in situ hybridization. The three genes were all expressed in 1 d~7d, distinct in some stages and weak in other stages.The expression of GH3^ Lmbrl and Shh varied with time. In Silkies, GH3 expression was higher in the heads and bodies (4d up to 7.18 times) than limb buds (4d up to 2.15 times) . In White Leghorns, GH3 expression in the limb buds after 4.5d rose rapidly up to 7.55 times, forming a sharp contrast to those of their heads and bodies (little variation , 5d up to 2.54 times) as well as three parts of the Silkies intensively. Repeated experiments got the same results. There has been no such similiar report until now. Lmbrl and Shh in limb buds of two breeds varied little with time, although Lmbrl reached higher (3.5d up to 6.27 times) in the heads and bodies of Silkies before 4.5d.The expression of three genes was also different between the two breeds. The expression of GH3 in the heads and limb buds was higher in 3d~4d and lower in 4.5d~6d in Silkies than that in White Leghorns. The expression of Shh in the limb buds was higher in Silkies than that in White Leghorns (except 6d), which is in reverse to that of the bodies. When GU3 rose rapidly, Shh descended quickly all the time, presenting antagonistic relationship. 4d~6d is the critical stage of toe development. At the very time, higher GH3 and lower Shh expression in the limb buds of White Leghorns might avoid forming 5-toed, while higher Shh and lower GH3 in the limb buds of Silkies might help to form 5-toed.The little expression difference of Lmbrl in two breeds and the SNPs in the introns of Lmbrl related to toes, Linking up other studies on Lmbrl in orientation, expression and variation in vertebrate, indicated that Lmbrl was a key gene in chicken toes development, likely to regulate toes growth by changing the binding sites of transcription factors.Chicken polydactyly is formed by extra Shh expression, which can be arose by the expression quantity of GH3 or intron variation of Lmbrl.
SNP搜寻所用实验材料共6组,有丝羽乌骨鸡(五趾)、A系肉鸡(四趾)、四趾菊花鸡、五趾菊花鸡、四趾北京油鸡和五趾北京油鸡。每组选5个个体对候选基因进行PCR后分6组混合回收产物并测序比对,在五个候选基因的外显子、内含子保守区以及Lmbr1的5’上游区寻找能严格区别四趾和五趾表型的SNPs作为趾型相关SNPs。结果显示这五个候选基因的外显子相当保守,只发现Lmbr1外显子16的一处C/T变异和Gli3外显子15的G/A变异,没有造成氨基酸改变,但测序比对或SSCP筛查排除了它们与趾型的关系。在候选基因的内含子保守区我们只发现Lmbr1的内含子5有一处C/A变异与趾型相关,定位在chr2:7901586bp。所有的四趾鸡该位点都为C,所有的五趾鸡该位点都为A/N。对菊花鸡、北京油鸡、CAU资源家系(由A系肉鸡和丝羽乌骨鸡建立)共517个体中进行了SSCP群体分型,证实了这个SNP的确与趾型相关。在Lmbr1 5’上游区段的筛查中,我们从26处变异中筛查出6处趾型相关SNP,并将这6处SNP定位在Chr2:7922655～Chr2:7928742bp之间。
在MatInspector网上预测中,Lmbr1 5’上游的6处SNP中有3处改变了同源异形蛋白因子或胚胎形成有关的蛋白结合位点。Lmbr1内含子5的变异至少改变了3个同源异形蛋白因子的结合位点,为其编码的Hox基因家族在人和鼠多趾的发育与模式形成中起着重要作用。预测结果也支持Lmbr1与趾型有关。
候选基因的表达研究采用白莱航(四趾)和丝羽乌骨鸡(五趾)的纯繁种蛋同批孵化,选择同期发育的胚胎分为头、躯体和肢芽三部分提取RNA。采用RT-PCR法,我们最早在1d(孵化24小时)检测到Gli3、Lmbr1和Shh的表达,早于胚胎原位杂
Polydactyly is a character in Several chicken breeds, which is a good material to study the interaction among genes in the process of limb growth for it is easy to operate and observate .Considering the interaction among polydactyly relevant genes and the polydactyly homologous region in human and mouse, we selected En2, Shh, Lmbr1 , Hlxb9 and Gli3 as polydactyly candidate genes in chicken. The SNPs related to toes and the difference in the genes expression were found and detected, in order to uncover the functions of candidate genes and explain the mechanism of chicken polydactyly..The materials used for finding SNPs included six groups:Silkies(5-toed), A strain of broilers (4-toed), Juhua chickens (4-teod and 5-teod) and Beijing-youji chickens (4-teod and 5-teod) .With 5 chickens from each group, exons and intron-conserved-domains (ICDs ) of five candidate genes, and 5' upstream of Lmbr1 were amplified separately. Then each sequence were purified and compared by 6 groups to search for the SNPs which could strictly distinguish 4-toed from 5-toed. The exons of the 5 genes were conservative and only two SNPs were found , one SNP (C/T) in exonl6 of Lmbr1 , the other SNP (G/A) in exon15 of Gli3. More sequences comparison and SSCP results exclude their relation to toes. But in ICDs, only one SNP(C/A) in Lmbrl intron5 was found and mapped to chr2:7901586bp, C variation correlating with 4-toed and A variation correlating with 5-toed, which was consistent with SSCP typing results from 517 DNAs of Juhua chickens, Beijing-youji chickens, CAU resource reference population(established by Silkies and A strain of broilers) .From the 5'-upstream of Lmbrl , 6 out of 26 SNPs were found related to toes, mapped to Chr2:7922655~ Chr2:7928742bp.Accordong to internet MatInspector prediction, 3 of the 6 SNPs could change homeodomain factor or protein binding sites that relevant to embryogenesis; the SNP in the intron5 of Lmbrl could change three homeodomain transcription factors binding sites. Their coding genes. Hox gene family, play important roles in the anterior-posterior axis in the development of polydactyly in human and mouse. The prediction results supported the influence of Lmbrl in the development of toes.To detect the differences in candidate genes' expression, RNAs were
used, which were extracted from heads, bodies and limb buds of White Leghon embryos (4-toed) and Silkie embryos (5-toed) at the same stage.The expression of GHS, Lmbrl and Shh was initially detected in Id (24h incubation) by RT-PCR, ahead of the first detectable time 2.5d~3d in Whole-mount in situ hybridization. The three genes were all expressed in 1 d~7d, distinct in some stages and weak in other stages.The expression of GH3^ Lmbrl and Shh varied with time. In Silkies, GH3 expression was higher in the heads and bodies (4d up to 7.18 times) than limb buds (4d up to 2.15 times) . In White Leghorns, GH3 expression in the limb buds after 4.5d rose rapidly up to 7.55 times, forming a sharp contrast to those of their heads and bodies (little variation , 5d up to 2.54 times) as well as three parts of the Silkies intensively. Repeated experiments got the same results. There has been no such similiar report until now. Lmbrl and Shh in limb buds of two breeds varied little with time, although Lmbrl reached higher (3.5d up to 6.27 times) in the heads and bodies of Silkies before 4.5d.The expression of three genes was also different between the two breeds. The expression of GH3 in the heads and limb buds was higher in 3d~4d and lower in 4.5d~6d in Silkies than that in White Leghorns. The expression of Shh in the limb buds was higher in Silkies than that in White Leghorns (except 6d), which is in reverse to that of the bodies. When GU3 rose rapidly, Shh descended quickly all the time, presenting antagonistic relationship. 4d~6d is the critical stage of toe development. At the very time, higher GH3 and lower Shh expression in the limb buds of White Leghorns might avoid forming 5-toed, while higher Shh and lower GH3 in the limb buds of Silkies might help to form 5-toed.The little expression difference of Lmbrl in two breeds and the SNPs in the introns of Lmbrl related to toes, Linking up other studies on Lmbrl in orientation, expression and variation in vertebrate, indicated that Lmbrl was a key gene in chicken toes development, likely to regulate toes growth by changing the binding sites of transcription factors.Chicken polydactyly is formed by extra Shh expression, which can be arose by the expression quantity of GH3 or intron variation of Lmbrl.
引文
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