大规模板栗疫病菌cDNA文库的特征和冗余序列的排除

低毒病毒/板栗疫病菌是一个研究病毒宿主相互作用的优良的实验系统.为了更好的了解病毒与宿主相互作用的机制,我们建立了一个野生型无病毒板栗疫病菌菌株EP155和受低毒病毒感染的菌株EP713的cDNA混和文库,以进行表达序列标签（EST）测定.对543个克隆的外源片段的分析表明,该文库克隆中含外源片段的比例为89.8%;通过小规模测序,发现空质粒与小片段克隆占测序总数的10.1%;表达丰度最高的4个克隆出现次数的总和占测序总数的9.6%.通过设计引物进行PCR和原位杂交,对cDNA文库2万多个克隆进行了筛选,共筛除了占总数19.7%的空质粒与小克隆和5.5%的高丰度克隆,为高效率的大规模EST测序准备了条件.

Characterization and discrimination of redundant sequence of a large-scale cDNA library of Cryphonectria parasitica

Hypovirus/Cryphonectria parasitica is an excellent experimental system to study the virus-host interaction.In order to have a better insight of the virus-host interaction,a mix cDNA library of C.parasitica was prepared from mRNA isolated from the wild-type strain EP155(virus-free) and the hypovirus-infected strain EP713.Based on the analyses of 543 clones,89.8% of the clones from the library were found to contain cDNA insert.Empty plasmids and small fragment clones accounting for 10.1% and four highly abundant sequences accounting for 9.6% were found through a small-scale sequencing.By PCR and in situ hybridization of more than 20 000 clones from the cDNA library,19.7% of the total clones screened were found to be insert-less or with only a small insert and 5.5% to be highly abundant sequences.This work resulted in a collection of high quality cDNA clones with significantly reduced redundancy that set a good start for the EST sequencing.