水貂ASIP基因单倍型检测及其皮肤组织mRNA差异表达分析

Haplotype Detection and mRNA Expression Level Analysis of ASIP Gene in Skin of American Mink (Neovison vison)

试验旨在探究鼠灰色(agouti signaling protein,ASIP)基因单核苷酸多态性(single nucleotide polymorphisms,SNPs)形成的单倍型及皮肤组织差异表达mRNA对水貂被毛色素沉积的影响。通过PCR扩增、Sanger测序技术对金州黑水貂、红眼白水貂和名威银蓝水貂ASIP基因进行SNPs单倍型检测分析,利用实时荧光定量PCR技术检测3种毛色皮肤组织ASIP基因的表达量,分析单倍型及mRNA差异表达与毛色表型的相关性。结果表明,301个样本中共检测到10个SNPs,内含子2中4个SNPs(G18A、A159G、G235T、C1189T)共形成10种单倍型(Hap1~Hap10),其中Hap1(GAGC)和Hap2(GAGT)是3种不同毛色水貂群体的共享单倍型;部分内含子3中6个SNPs(C252T、A290C、G298C、A340G、T343C、T379C)形成4种单倍型(Hap1~Hap4),且Hap2(CCCGCC)是名威银蓝水貂群体的主体单倍型。5个位点(A290C、G298C、A340G、T343C、T379C)均处于完全连锁不平衡状态。实时荧光定量PCR检测显示,金州黑水貂和名威银蓝水貂ASIP基因mRNA表达量分别是红眼白水貂的1.25和0.95倍,三者间差异不显著(P>0.05)。研究结果初步提示,ASIP基因调控水貂不同毛色表型形成的分子机制可能存在差异。

The aim of this study was to explore the effects of haplotype formed by single nucleotide polymorphisms(SNPs)of agouti signaling protein(ASIP)gene and analyze the association of mRNA differential expression with coat color phenotype in American mink(Neovison vison).The SNPs haplotypes of ASIP gene in Jinzhou Black mink,Red eye White mink and Mingwei Silverblue mink were detected by PCR and Sanger direct sequencing technology.The mRNA expression levels of ASIP gene in skin tissue with three kinds of coat color were detected by Real-time PCR,and then the correlation between haplotypes,mRNA differential expression levels and coat color traits were analyzed.The results showed that a total of 10 SNPs were detected in 301 samples,of which 4 SNPs(G18A,A159G,G235T and C1189T)were located in intron 2,6 SNPs(C252T,A290C,G298C,A340G,T343C and T379C)were located in partial intron 3.10(Hap1-Hap10)and 4(Hap1-Hap4)haplotypes were formed by 4 and 6 SNPs,respectively,of which Hap1(GAGC)and Hap2(GAGT)were shared haplotypes in intron 2,Hap2(CCCGCC)was the main haplotype in Mingwei Silverblue mink.5 loci(A290C,G298C,A340G,T343C and T379C)were in complete linkage disequilibrium.Real-time PCR results showed that the mRNA expression levels of ASIP gene of Jinzhou Black mink and Mingwei Silverblue mink were 1.25 and 0.95 times,which were higher than that of Red eye White mink,there was no significant difference among three coat colors(P>0.05).The results indicated that the molecular mechanism of ASIP gene regulating the formation of mink coat color phenotype might be different.