【作者】 陈赛娟；

【导师】 谷子林；

【作者基本信息】 河北农业大学 ， 动物遗传育种与繁殖， 2011， 博士

【摘要】 獭兔(力克斯兔)是一种典型的裘皮用兔,评价獭兔毛皮品质的主要指标有被毛密度、被毛长度、粗毛率、绒毛细度和皮张面积,而目前我国獭兔毛皮品质方面存在的问题主要集中在被毛密度及皮张面积上。如何提高我国獭兔的毛皮品质以提高市场竞争力,是目前亟待解决的问题。本研究以獭兔为研究对象,利用基因芯片技术,对不同被毛密度獭兔的皮肤组织中差异表达的基因进行筛选,并采用实时定量PCR方法对芯片结果进行验证;采用RT-PCR方法克隆獭兔的CCNA2基因序列,通过测序结合PCR-RFLP技术对獭兔CCNA2基因进行单核苷酸多态性检测,并探讨其与獭兔毛皮品质特别是被毛密度的关系,旨在为獭兔分子标记辅助选择提供有效的遗传标记。研究结果如下:⑴利用家兔全基因表达谱芯片筛选出了2 657个在不同被毛密度獭兔皮肤组织间差异表达的基因,其中包含1 106个已知功能的基因,表达上调的基因687个,表达下调的基因419个。GO分类显示:大部分差异表达的基因位于细胞膜、细胞融入膜或细胞融入质膜;属于核苷酸、蛋白质、DNA及离子结合类分子;主要涉及细胞信号转导,发育以及细胞增殖、分化、凋亡等。KEGG信号通路分析显示:在表达上调的基因中95个通路功能状态发生的改变有统计学意义(P<0.05),涉及细胞粘附分子、TGFβ、MAPK、Wnt信号通路等;在表达下调的基因中87个通路功能状态发生的改变有统计学意义(P<0.05),涉及细胞周期、氧化磷酸化、p53信号通路等。实时定量PCR结果与基因芯片结果基本吻合,证实基因芯片结果可靠。⑵通过基因芯片试验,很多与细胞增殖、分化、凋亡、细胞信号转导等有关的基因被筛选出来,如:MMP2、TGFβ1、TGFβ2、IGF-1B、BMP2、ActRⅡB、CDK2、CCNA2等,这些基因的差异表达,可能参与皮肤毛囊的形成或分化过程,最终导致獭兔被毛密度的差异。结合多方面的分析结果,最终选定细胞周期素A2(CCNA2)基因作为后续试验的候选基因。⑶通过RT-PCR方法克隆出了獭兔的CCNA2基因序列。通过测序结合PCR-RFLP技术检测出了CCNA2基因的两个SNPs位点:129G>A位点引起了限制性核酸内切酶位点ScaⅠ的变化,是丙氨酸的同义突变;1140G>C位点引起了限制性核酸内切酶位点AfIⅡ的变化,是从赖氨酸到天冬酰胺的错义突变。⑷在试验兔群中,CCNA2基因的129G>A位点的G等位基因为优势基因,GG型的基因型频率高于AA型的基因型频率;1140G>C位点的G等位基因为优势基因,GG型的基因型频率高于CC型的基因型频率。这两个位点均处于中度多态(0.25<PIC<0.5),遗传变异较大;均处于Hardy-Weinberg平衡状态(P>0.05)。⑸对獭兔的CCNA2基因多态性和毛皮品质相关性分析表明,129G>A位点的GG基因型的背中部被毛密度显著高于GA型和AA型;1140G>C位点的GG基因型的背中部被毛密度显著高于GC型和CC型;CCNA2基因129G>A位点和1140G>C位点的GG基因型均能在不影响獭兔皮张面积、被毛长度和绒毛细度的情况下,显著增加其被毛密度。可以将其作为影响獭兔毛皮品质特别是被毛密度的分子标记。更多还原

【Abstract】 Rex rabbit is a typical rabbit for fur. Wool density, hair length, coarse rate, villus diameter and area of hide are main indices of fur quality. In china, the current problem of rabbit fur quality is mainly focusing on the wool density and area of hide. How to improve the quality of our rex rabbit’s fur in order to enhance market competitiveness is a critical problem. In this study, the gene expression patterns in different wool density of rex rabbit are presented and analyzed with cDNA microarray and statistical analysis, then verify the result of selected genes from microarray experiments by qRT-PCR. In order to give the effective molecular markers for the marker-assisted selection (MAS) on early production performance of rex rabbits, SNPs polymorphism of CCNA2 gene were conducted on rex rabbit by PCR-RFLP and DNA sequencing method. The relationship between SNPs polymorphism and fur traits had also been analyzed.The results were as follows:⑴The 2657 differentially expressed genes were identified in different wool density of rex rabbit. Among them, 1103 genes were functionally known genes. 687 genes were up-regulated and 419 down-regulated. GO analysis indicated that most differently expressed genes were monitored to be relative with cell signal transduction, cell development and cell proliferation, cell division, apoptosis, which were mainly belong to nucleotide binding, protein binding, DNA binding and ion binding molecule. And these genes were usually located at cell envelope. KEGG analysis showed that 95 signal pathways associated with up-regulated genes had changed significantly (P<0.05), these pathways were involved in focal adhesion, TGF-beta signaling pathway, cell adhesion molecules, MAPK signaling pathway, Wnt signaling pathway and so on; 87 signal pathways associated with down-regulated genes had changed significantly (P<0.05), this pathways were involved in cell cycle, oxidative phosphorylation, regulation of actin cytoskeleton, p53 signaling pathway and so on. The alteration of gene expression from array data was confirmed by quantitative real-time PCR.⑵Some important differentially expressed genes in different wool density of rex rabbit were identified, such as MMP2, TGF-β1, TGF-β2, IGF-1, BMP2, ActRⅡB, CDK2 and CCNA2. The abnormal expression of this genes may play an important role in the hair follicles initiation and development, finally lead to the distinction in wool density of rex rabbit. After bioinformatics analysis for all differently expressed genes, CCNA2 gene was selected as candidate gene in future studies.⑶Sequences of CCNA2 gene were amplified from the genome RNA samples extracted from blood of rex rabbits by RT-PCR. The polymorphisms of CCNA2 gene were detected by DNA sequencing and PCR-RFLP methods. The 129G>A SNP is a synonymous mutation of alanine, which creates the ScaⅠrestriction site. The 1140G>C SNP is a missense mutation from lysine to asparagine, which destroies the AfIⅡrestriction site.⑷At the 129G>A and 1140G>C SNP marker, the GG genotypes were dominance in population of rex rabbit, respectively; the G alleles were dominant allele, respectively. The two polymorphism sites showed moderate polymorphism(0.25<PIC<0.5), and were all in Hardy-Weinberg equilibrium(P>0.05).⑸The association between the CCNA2 polymorphism and fur traits (body weight, wool density, wool fineness and hair length) were analyzed in Rex rabbit. At the 129G>A SNP marker, the wool density of the GG genotype are significantly higher than those of GA and AA genotype; At the 1140G>C SNP marker, the wool density of the GG genotype are significantly higher than those of GC and CC genotype. The results indicated that, at the 129G>A and 1140G>C SNP marker, the GG genotype have high wool density, and have no adverse effect on the other fur indices(hair length, wool fineness and body weight). Therefore, the GG genotypes of“129G>A”and“1140G>C”were the most favorable at the CCNA2 gene for wool density trait in Rex rabbit.更多还原