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朝鲜鹌鹑OCA2基因克隆及其多态性与羽色的相关性
刘 佩1, 庞有志2, 张小辉,等2
1.河南科技大学 动物科技学院;2.河南科技大学 动物科技学院,洛阳市动物遗传繁育重点实验室
摘要:
【目的】克隆朝鲜鹌鹑(Korean quail)OCA2基因的cDNA序列,检测其在不同羽色(白羽和栗羽)群体胚胎发育不同阶段翅组织中mRNA的表达情况,探讨OCA2基因多态性对朝鲜鹌鹑羽色的影响,筛选可能与羽色相关的多态性位点。【方法】取白羽、栗羽朝鲜鹌鹑孵化6,8,10,12和14 d的胚胎翅组织,提取总RNA,RT-PCR扩增OCA2基因cDNA序列,利用实时荧光定量PCR检测OCA2基因mRNA在朝鲜鹌鹑胚胎发育不同阶段和不同羽色鹌鹑胚胎翅组织中的表达水平;用胚胎发育10 d时的cDNA样品扩增OCA2基因cDNA序列并进行生物信息学分析。以300羽白羽朝鲜鹌鹑和栗羽朝鲜鹌鹑孵化10 d时的胚胎为研究材料,采集翅组织样品,提取基因组DNA, PCR扩增OCA2基因,通过不对称PCR SSCP方法结合测序确定基因型,分析不同基因型对朝鲜鹌鹑羽色形成的影响。【结果】朝鲜鹌鹑OCA2基因胚胎发育6~8 d时的表达量低于胚胎发育10~14 d时的表达量,且在10 d时达到最高。6~8 d时,OCA2基因的表达水平在2种羽色朝鲜鹌鹑胚胎中差异不显著;10~14 d时,栗羽朝鲜鹌鹑OCA2基因mRNA的表达水平显著高于白羽朝鲜鹌鹑(P<0.05)。OCA2基因CDS区片段大小为2 660 bp,含有24个外显子,开放阅读框长2 556 bp,编码含851个氨基酸的稳定疏水性蛋白。在朝鲜鹌鹑OCA2基因中检测到3个SNP位点,分别为外显子2上的c.93A>G、外显子9上的c.999A>G、3′UTR上的c.2569C>T;群体遗传学分析显示,3个突变位点均表现为中度多态(0.25<PIC<0.5),χ2检验表明朝鲜鹌鹑群体在3个SNP位点上均处于Hardy Weinberg平衡(P>0.05)。在3′UTR上的c.2569C>T位点,栗羽朝鲜鹌鹑3种基因型(CC、 CT和TT)的频率分布与白羽朝鲜鹌鹑存在极显著差异(P<0.01),说明该突变位点与鹌鹑羽色之间存在一定的关联性。【结论】白羽朝鲜鹌鹑翅组织中OCA2基因低表达可能与其羽毛白化有一定的关系,OCA2基因3′UTR上的c.2569C>T突变与朝鲜鹌鹑羽色性状之间具有一定的关联性,可以作为研究朝鲜鹌鹑羽色的候选基因。
关键词:  朝鲜鹌鹑  OCA2基因  羽色  基因多态性
DOI:
分类号:
基金项目:河南省产学研合作项目“蛋用鹌鹑自别雌雄配套系的培育与产业化”(152107000098.0)
Cloning analysis of OCA2 gene relationship of polymorphism with plumage color in Korean quail (Coturnix coturnix)
LIU Pei,PANG Youzhi,ZHANG Xiaohui,et al
Abstract:
【Objective】The main purpose of this study was to clone OCA2 gene of Korean quail and analyze its expression levels in wing tissues at different embryo developmental stages of maroon Korean quail and white Korean quail.The association of single nucleotide polymorphism (SNP) of OCA2 gene with the plumage color of Korean quail was also studied.【Method】The total RNA was extracted from wing tissues of Korean quail embryos at 6,8,10,12 and 14 d of incubation,the cDNA fragment of OCA2 gene was amplified by RT-PCR,and the expression levels of OCA2 gene mRNA were detected in different developmental stages using real-time quantity PCR.The cDNA fragment of OCA2 gene was amplified using cDNA of wing samples at 10 d of incubation and the sequences were analyzed by bioinformatics methods.Genome DNA was extracted from wing tissues of 300 Korean quail embryos at 10 d of incubation and genotypes were detected using asymmetric PCR-SSCP and DNA sequencing. The effects of different genotypes on plumage color of Korean quail were analyzed.【Result】The expression levels of OCA2 mRNA were lower at 6-8 d than that at 10-14 d,then the expression of OCA2 gene mRNA reached the peak at 10 d.The expression levels of OCA2 mRNA had no significant difference between maroon Korean quail and white Korean quail embryos at 6-8 d.The expression levels of OCA2 mRNA in maroon Korean quail embryos were significantly higher (P<0.05) than in white Korean quail embryos at 10-14 d of incubation.The obtained CDS fragment of OCA2 gene was 2 660 bp,including 24 exons.The open reading frame (ORF) of 2 559 bp encoded stable hydrophobic protein consisting of 851 amino acids.A total of 3 SNPs (c.93A>G in exon 2,c.999A>G in exon 9,and c.2569C>T in 3′UTR) were detected in OCA2 gene of Korean quail and all of them had moderate polymorphisms (0.25<PIC<0.5) by population genetic analysis and stayed in Hardy Weinberg equilibrium (P>0.05) by χ2 test.The distributions of three genotypes (CC,CT and TT) c.2569C>T in 3′UTR in maroon Korean quail were significantly different with that in white Korean quail,indicating that this SNP had strong association with plumage color in Korean quail (P<0.01).【Conclusion】The low expression level of OCA2 mRNA in wing tissue may be related to the albinistic feather in white Korean quail,and the mutation (c.2569C>T) in 3′UTR of OCA2 gene had strong association with feather color in Korean quail.The OCA2gene could be regarded as a candidate gene of feather color regulation in Korean quail.
Key words:  Korean quail  OCA2 gene  feather color  gene polymorphism

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