【作者】 苏蕊；

【导师】 李金泉； 张文广；

【作者基本信息】 内蒙古农业大学 ， 动物遗传育种与繁殖， 2009， 博士

【摘要】 绒山羊皮肤毛囊基因表达的模式对绒毛产量和品质有重要的作用,探讨绒山羊绒山羊绒毛生长、脱落时期皮肤基因表达模式的特点对绒山羊的选育具有重要意义。本研究通过功能分类基因芯片对内蒙古绒山羊绒毛生长、脱落时期皮肤基因的表达进行了检测,利用生物信息学的方法对上、下调的基因进行了归纳和分析;对用上、下调最明显的BMP2基因进行了基因芯片的验证,同时利用组织切片原位杂交方法对该基因在毛囊的定位表达进行了分析。结果如下:1、采用包含288个基因的基因芯片对3月份初生羔羊及5月份成年羊皮肤检测对比结果表明,绒毛脱落期的成年绒山羊羊(5月份成年羊)同绒毛生长期的初生羔羊(3月份初生羔羊)相比,共有8个基因表达上调,占全部基因的3%;共有25个基因表达下调,占全部基因的9.5%。2、采用包含288个基因的基因芯片对5月份成年绒山羊及9月份成年绒山羊皮肤检测对比结果表明,绒毛生长期的成年绒山羊羊(9月份成年羊)同绒毛休止期的成年绒山羊(5月份成年羊)相比,共有4个基因表达上调,占全部基因的1.5%;共有31个基因表达下调,占全部基因的11.8%。3、采用包含288个基因的基因芯片对对3月份初生羔羊及9月份成年羊皮肤检测对比结果表明,绒毛生长期的成年绒山羊羊(9月份成年羊)同绒毛生长期的初生羔羊(3月份初生羔羊)相比,共有5个基因表达上调,占全部基因的1.9%;共有44个基因表达下调,占全部基因的16.8%。4、通过生物信息学分析得出,在下一步的生物学实验中,可将POU3F3、POU5F1、UTF1、KGF、Integrinα6、Trk C及BMP2作为重要的候选基因进行分析。5、利用GO数据库对基因芯片杂交结果统计分析得出:在绒山羊绒毛生长、脱落时期,初生羔羊及成年羊的相关基因表达具有特异性,且差异表达的基因主要为涉及信号转导及细胞发育类的分子,这些分子以核苷酸结合、蛋白结合、DNA结合或离子结合类分子为主,主要分布于细胞膜、细胞融入膜或细胞融入质膜上。6、对BMP2基因在内蒙古绒山羊绒毛生长、脱落时期皮肤及毛囊中表达检测的结果表明:BMP2基因在绒山羊绒毛脱落期高表达,是绒毛生长期该基因表达量的27.8倍;原位杂交表明该基因在绒毛脱落期次级毛囊的毛干周围强烈表达,而在兴盛期没有检测到阳性信号。7、通过不同时期、不同类型皮肤基因表达模式的特点,认为在今后绒山羊毛囊发育分子调控的研究中,可将研究时期固定为毛囊发育休止期,即绒山羊绒毛脱落期,并且可将在这个时期明显高表达的BMP2基因作为参照基因进行分析研究。更多还原

【Abstract】 The quantity and quality of cashmere are associated mainly with the gene expression mode of skin. So it is crucial to character the mode of gene expression in skin in anagen and telogen.in this study, we analyze the gene expression profile of Cashmere goat skin in anagen and telogen via Functional Classification Microarray. We chose BMP2 for real-time PCR to confirm our result. The results agree with the microarray data. Finally, we investigated the expression of BMP2 gene during anagen and telogen by situ hybridization. The results suggested as following:1. Analyzed transcriptional differences between adult goats on May and newborn goats on March at gene expression level. Compared with the newborn goats on March, 33 different genes of total 288 genes on the array were differentially two-fold or greater. Among these 33 genes differentially expressed, 8 genes were up-regulated, the percentage was 3%, whereas 25 genes were down-regulated with the percentage 9.5%.2. Analyzed transcriptional differences between adult goats adult goats on May and adult goats on September at gene expression level. Compared with the adult goats on September, 35 different genes of total 288 genes on the array were differentially two-fold or greater. Among these 35 genes differentially expressed, 4 genes were up-regulated, the percentage was 1.5%, whereas 31 genes were down-regulated with the percentage 11.8%.3. Analyzed transcriptional differences between newborn goats on March and adult goats on September at gene expression level. Compared with the adult goats on September, 49 different genes of total 288 genes on the array were differentially two-fold or greater. Among these 49 genes differentially expressed, 5 genes were up-regulated, the percentage was 1.9%, whereas 44 genes were down-regulated with the percentage 16.8%.4. After bioinformatics analysis for all genes differently expressed two-fold or more above, 6 genes associated with hair follicle development were found: POU3F3, POU5F1, UTF1, KGF, Integrinα6, Trk C and BMP2, which can be recognized as candidate gene in future studies.5. Bioinformatics analysis results reveal that most differently expressed genes were monitored to be relative with cell signal transduction and cell development, which were mainly belong to nucleotide binding, protein binding, DNA binding and ion binding molecule. And these genes were usually located at cell envelope. 6. Real-time quantitative PCR was employed to quantify the BMP2 expression in adult Inner Mongolia Cashmere goat skin. The expression of BMP2 in telogen was 27.8 times greater then expression during anagen phase. No significant change in the level of the expression ofβ-actin (house-keeping gene) was observed at anagen phases. Results of in situ hybridization indicated that BMP2 expressed at hair shaft in secondary hair follicle in telogen. However, it was found to be absent in anagen.7. Telogen could be recognized as the reference phase and BMP2 gene could be recognized as the reference gene in hair follicle development in future study.更多还原