【作者】 周兵；

【导师】 刘益平； 何大乾；

【作者基本信息】 四川农业大学 ， 动物遗传育种与繁殖， 2010， 硕士

【摘要】 毛色/羽色是一种可利用的经济性状和遗传标记,它在确定品种纯度、杂交组合和亲缘关系方面具有重要作用。黑素皮质素受体1(Melanocortin 1 Receptor, MClR)基因变异与许多脊椎动物(包括一些鸟类)的毛色/羽色表型相关,是毛·色/羽色遗传的重要候选基因。本研究以MClR基因为研究对象,采用PCR产物直接测序的方法,对朗德鹅灰羽和花羽(灰白相间)、浙东白鹅白羽3个羽色系MClR基因的部分编码区进行了单核苷酸多态性(Single Nucleotide Polymorphism, SNP)筛查和遗传多样性分析,并通过荧光定量PCR技术,分析MClR基因在1d、14d、21d、28d、42d和56d六个时间点品种间的表达差异和在皮肤组织中不同时间点的发育性变化,以探讨MClR基因变异对浙东白鹅和朗德鹅羽色遗传的影响,从而为遗传育种研究提供分子生物学依据。本试验扩增了浙东白鹅和朗德鹅MClR基因编码区的714bp。通过生物信息学分析,发现A+T含量为40.20%,G+C含量为59.52%,A+T含量远低于G+C含量。浙东白鹅与朗德鹅MClR基因与GenBank中其它雁属MClR基因的核苷酸同源性高达99%以上。通过对SNP位点的筛选,在所有群体中共筛查到5个SNPs,即210C>T、321C>T、411 C>T、525C>T、756G>A,且都为简约信息位点。其中210C>T、321C>T、756G>A3个突变位点只存在于浙东白鹅中,411C>T、525C>T 2个突变位点只存在于朗德鹅中,经过与GenBank中已提交的鹅的MClR基因序列比对,发现这5个SNPs都是在浙东白鹅和朗德鹅中新发现的SNPs。将核苷酸序列翻译后发现,这5个突变位点并未引起氨基酸序列的改变,均为同义突变。用Phase软件进行分型,共分出7种单倍型,其中单倍型Z2(C-C-C-C-G)和Z4 (C-C-T-C-G)为优势单倍型,Z2Z2和Z4Z4单倍型组合为优势单倍型组合。经卡方独立性检验,这5个突变位点不同基因型和单倍型在鹅品种间和羽色性状间的分布差异都达到了极显著水平的水平,表明MClR基因对鹅的羽色性状有着重要影响,说明MClR基因是鹅羽色的主要候选基因。本研究采用荧光定量PCR技术检测了MClR基因mRNA水平表达情况,结果表明,MClR基因在浙东白鹅1d皮肤组织中的表达量最高,而在朗德鹅中却是在28d皮肤组织中表达量最高。对于同一时间点,除1d和21d外,其余时间点浙东白鹅和朗德鹅品种间的表达差异均达到了显著或极显著的水平。对于同一品种不同时间点而言,浙东白鹅1d与其他日龄之间的表达差异达到了显著或极显著水平,朗德鹅28d与其他日龄(除14d外)之间的表达差异达到了显著或极显著水平。这些表达规律与品种和换毛有一定的关系。更多还原

【Abstract】 Coat color is available economic traits and genetic markers, it is in determining the species purity, and relationship crosses play an important role. Sequence variation in the melanocortin-1 receptor gene (MCIR) is associated with melanistic phenotypes in many vertebrate taxa, including several avian species. Consequently, the MClR gene is a candidated gene for melanin trait of many animals. In our study, the gray plumage Landes, the variegated plumage Landes (gray and white), and the White plumage Zhedong White Goose’s MClR gene genetic variation were studied with the PCR product direct sequencing method. The MClR gene expression was studied by fluorescence quantitative PCR reaction.We sequenced 714 bp of the MClR coding region in 176 individuals spanning the range of plumage variation. Through bioinformatics analysis, we found the A T content of the obtained sequence was 40.20%, the GC content was 59.52%, the AT content is much lower than GC. The homology of the obtained sequence from samples and partial MClR gene of geese was as high as 99%.By screening single nucleotide polymorphism sites in all individuals, we found five SNPs sites,210C>T,321C>T,411C>T,525C>T,756G>A, all of them were singleton variable sites. Three mutations,210C>T,321C>T,756G>A were only in the Zhedong White Goose, two variation sites,411C>T,525C>T were only in Landes. After the sequences alignment, we found that all the five SNPs sites in Zhedong White Goose and Landes were novel SNPs, but all were synonymous mutations. The results of alignment showed seven haplotypes, haplotype Z2 (C-C-C-C-G) and Z4 (C-C-T-C-G) were the dominant haplotype, Z2Z2 and Z4Z4 haplotype combination were the dominant haplotype combinations. The chi-square test results showed that the five mutations in different haplotypes and geese have reached highly significant level, indicating that MClR gene have a significant impact in geese plumage traits, suggesting that MClR gene is the main candidate genes for geese plumage traits.In order to detect the expression of MClR gene in mRNA level, quatitative PCR was conducted, which showed that MClR gene has the highest expression in 1 day-old skin Zhedong White Goose, while the Landes’ was in 28 day. For the same time, the expression difference in all the time points reached significant or highly significant level except the 1 day and 21 days between Zhedong White Geese and Landes Geese. For the same species at different time points, the expression difference between 1 day and other days reached significant or highly significant in Zhedong White Goose, Landes 28 days between other days (except 14 days) expression difference reach the significant or highly significant. The expression pattern may be related to species and molt to a certain extent.更多还原