朝鲜鹌鹑GNAS基因表达、克隆及其多态性与羽色的相关性

Relationship of plumage color with expression and polymorphism of GNAS gene in Korean quail

为了研究朝鲜鹌鹑GNAS基因表达和多态性与羽色的相关性,取栗羽和白羽朝鲜鹌鹑不同发育时期胚胎翅尖组织,采用Trizol法提取总RNA,实时荧光定量PCR检测GNAS基因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mRNA的表达水平;克隆GNAS基因的CDS全序列并进行生物信息学分析;采集孵化至10 d胚胎翅尖组织提取基因组DNA,PCR扩增GNAS基因的特定序列,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。结果表明,胚胎发育8~14 d时,GNAS基因在栗羽鹌鹑胚胎组织中的表达水平极显著(P<0.01)高于白羽鹌鹑胚胎。GNAS基因CDS区开放阅读框长1140 bp,含有14个外显子,编码含379个氨基酸的稳定性亲水蛋白。在朝鲜鹌鹑GNAS基因中检测到2个SNP位点,分别为外显子12区域的g.119221T>A和3′UTR区域的g.121181A>G。栗羽朝鲜鹌鹑的3种基因型(AA、AB和BB)的频率分布与白羽朝鲜鹌鹑存在极显著差异(P<0.01)。结论表明,朝鲜鹌鹑组织中GNAS基因的表达量高低与其羽色的形成有一定的关系,GNAS基因3′UTR上的g.121181A>G位点突变与朝鲜鹌鹑羽色性状之间具有显著关联性,可作为研究朝鲜鹌鹑羽色的一个候选基因。

The present study was undertaken to explore the correlation between GNAS gene expression,polymorphism and plumage color in Korean quail.Trizol method was used to extract total RNA from different wing tip tissues of maroon Korean quail embryos and white Korean quail embryos at different development stages,and RT-qPCR was used to detect the mRNA expression level of GNAS gene in wing tissues of Korean quail embryos at different development stages and different feather colors;The full CDS sequence of the GNAS gene was cloned and analyzed by bioinformatics;Genomic DNA was extracted from wing tip tissue of embryos,incubated for 10 days,and the specific sequence of the GNAS gene was amplified by PCR.Asymmetrical PCR-SSCP method combined with sequencing was used to determine the genotype to analyze the effect of different genotypes on the formation of plumage color in Korean quail.The results showed that the expression level of GNAS gene in maroon quail embryos was significantly higher(P<0.01)than that in white quail embryos at 8-14 days.The open reading frame of the CDS region of the GNAS gene is 1140 bp in length,contains 14 exons,and encodes a stable hydrophilic protein containing 379 amino acids.Two SNP loci were detected in the Korean quail GNAS gene,namely g.119221T>A in exon 12 region and g.121181A>G in 3′UTR region.The frequency distribution of the three genotypes(AA,AB and BB)of maroon plumage Korean quail in g.121181A>G mutation site in 3′UTR region was significantly different from that of white plumage Korean quail(P<0.01).These results suggest that the expression level of GNAS gene in Korean quail tissue was related to the formation of plumage color.The g.121181A>G mutation on the 3′UTR of the GNAS gene had a significant correlation with the plumage color traits of Korean quail.GNAS can be used as a candidate gene for studying plumage color of Korean quail.