【作者】 何培彦；

【导师】 吴雪昌；

【作者基本信息】 浙江大学 ， 微生物， 2006， 硕士

【摘要】 原生质体融合技术可把优良菌株系谱中筛选获得的不同“正突变株”作为亲本，通过交换、重组，使优良性状集中于融合子，加快育种的速度，是一种极为重要的育种方法。但是为了便于检出融合子，融合亲株需要携带营养缺陷等筛选标记，而损伤亲株的遗传背景，使融合子的优良性状有所弱化。本文尝试使用RAPD标记筛选融合子，以实现酿酒酵母亲株无损伤融合子的构建。 本文用80个随机引物对欲作为构建融合子的酿酒酵母(S．cerevisiae)亲本菌株C18、C19基因组DNA多态性进行了RAPD分析。其中5个引物可在两亲本菌株间产生清晰且重复性好的特异性条带，获得了6条可以作为筛选标记的特异性谱带，其中3条属于C18，3条属于C19。经原生质体融合，构建了遗传背景无损伤的酒精发酵高转化率的一系列融合子，经过RAPD鉴定，其中的18株融合子其扩增图谱中不仅具有双亲共有的扩增条带，而且还具有双亲的特异性条带，证明其确为C18和C19的杂种。该18株融合子酒精发酵能力经与传统的用营养缺陷型标记筛选的23株融合子比较，表明用RAPD标记筛选到的融合子群体中，高产菌株所占的比例明显高于前者。因此，采用遗传背景无损伤的RAPD标记筛选融合子不但是可行的，而且对提高原生质体融合育种的效率有重要的意义。更多还原

【Abstract】 Protoplast fusion had become an important tool for breeding as it could quicken the process of breeding by concentrating valuable traits of two parental strains into one strain through transformation and recombination.In order to selected hybrids, selected markers such as auxotroph marker was always added to parental strains. That will damage the genetic background of parental strains and lessen valuable traits of fusants. In this paper, we attempted to construct fusants without damaging the genetic background of parental strains by selecting fusants by RAPD marker,We used 80 arbitrary primers to analyze the polymorphism of the genomic DNA of the two parental strains C18 and C19 in S.cerevisiae. 5 primers can amplify clear and reproducible polymorphic bands in two parental strains and produce 6 polymorphic bands that can regarded as selected marker, 3 specific to C18 and 3 specific to C19, and then C18 fused with C19 by protoplast fusion, we obtained a series of fusants without damaging the genetic background of parental strains and identified them by RAPD marker. Their RAPD profiles of 18 fusants not only contained common bands of parental strains, but also specific bands of parental strains, so they were hybrids of C18 and C19. Compared ethanol fermentation power of these 18 fusants with 23 fusants selected by auxotrophic marker, the results demonstrated that percentage of high ethanol-yielding strains in fusants population selected by RAPD marker was significantly high than fusants population selected by auxotrophic marker. So RAPD marker was suitable for selecting fusants. If it used in the field of strain breeding, it could improve the efficiency of strain breeding.更多还原