黄麻单染色体的分离和扩增及cDNA探针高通量筛选初探-生物化学与分子生物学专业毕业论文.docxVIP

摘要微分离微克隆技术对植物的遗传研究具有重要的应用价值。但是，由 摘要 微分离微克隆技术对植物的遗传研究具有重要的应用价值。但是，由 于大多数植物的染色体都很小且形态上难以识别，因而该技术在植物中的应 用很少。本研究以具有典型小染色体的植物黄麻为模式，对单染色体的分离 和体外扩增以及利用单染色体未克隆DNA文库和eDNA膜阵列高通量地筛 选特定染色体的特异eDNA探针进行了探讨。主要结果如下： 11用玻璃针显微分离法成功分离到了长果种黄麻(Corchorus olitorius ￡)闽引一号和圆果种黄麻(Corchorus capsularis L．)高雄青皮单染色体各 80条。 21将分离到的闽引一号和高雄青皮单染色体分别进行LA—PCR和 DOP．PCR两种方法的两轮扩增，分别得到250．2000bp和1 00—600bp之间的 DNA片段。用LA．PCR共扩增闽引一号和高雄青皮单染色体各45条：用 DOP．PCR共扩增闽引一号和高雄青皮单染色体各5条，从而建立了闽引一 号和高雄青皮单染色体未克隆文库各50个。Southern杂交表明，获得的扩 增片段确实来自黄麻基因组。 3)分别构建了闽引一号和高雄青皮的eDNA文库，两个文库的滴度 均达到了1．8-1．9×106个pfu／ml。 4)用同位素标记单染色体未克隆DNA文库，运用eDNA膜阵列杂交进 行高通量探针筛选的探索。结果预示了应用eDNA膜阵列高效率筛选特定染 色体特异探针的可行性。这一探索性研究将对黄麻单染色体的分类和建立高 密度分子标记连锁图谱及染色体进化和比较基因组研究奠定基础a 关键词：黄麻，微分离，eDNA阵列，探针 AbstractThe Abstract The technology of ellromosome microdissection and microcloning is useful for genetic research in plants．However,as most plants have small chromosomes that are difficult to be distinguished，the technology has not been widely applied to plants．In the pmsem studywith jute as a model，which has typical small chromosomes，the isolation and amplification of single chromosomes and the high·throughput screening of c11romosome—specific eDNA probes using uncloned DNA libraries of single c111．omosomes and eDNA macroarray were investigated． Major results are as follows： I)Single cllromosomes of Corchorus olitorius L Minyin NO．1 and Corchorus capsularis L．Gaoxiongqingpi were successfully isolated by micromanipulation with glass needles．80 chromosomes were obtained for each species． 2)The isolated jute c}lromommes were amplified by LA—PCR or DOP-PCR． After two rounds of PeR amplification，the DNA fragments ranged from 250bp to 2000bp were obtained by LA—PCR and 100bp to 600bp were obtained by DOP．PCR．45 chromosomes were amplified by LA-PCR and 5 chromosomes were amplified by DOP—PCR for Minyin NO．1 and Gaoxiongqingpi．Therefore， 50 uncloned single·chromosome DNA libraries were established for both Minyin NO．1 and Gaoxiongqingpi．Southern blot verified that the PCR produc