橡胶树甲基化过滤文库的构建

Construction of a methylation filtration library in Hevea brasiliensis

为高通量富集橡胶树基因编码序列,文章首次利用大肠杆菌McrBC限制修饰系统构建了巴西橡胶树基因组甲基化过滤文库。该文库未扩增和扩增后的滴度分别为2.6×106pfu/mL和9×109pfu/mL,阳性克隆率为86.4%,插入片段大小为1～2.5kb,平均长度为1.2kb。随机挑选100个克隆进行测序,共拼接出81个非冗余序列,其中包括6个重叠群(Contigs)和75个单一序列(Singlets),冗余度为17.35%。Blast分析发现,39.5%非冗余序列与Nr库中功能基因同源,14.81%与EST数据库中序列同源,32.1%序列为未知序列,且部分序列为开花调控、抗虫和抗病等功能相关的同源基因。橡胶树甲基化文库的建立为橡胶树重要功能基因发现和克隆提供一条重要途径。

In order to enrich gene encoding region of Hevea brasiliensis,a methylation filtration library was constructed using Escherichia coli McrBC restriction-modification system.The titers of the non-amplified library and the amplified library were 2.6×10^6 pfu/ml and 9.0×10^9,respectively.The rate of positive clones was 86.4%.The lengths of inserted DNA sequence ranged from 1 kb to 2.5 kb and the average size of inserts was 1.2 kb.One hundred clones were selected randomly for sequencing,resulting in splicing out of 81 non-redundant sequences,including 6 contigs and 75 singlets.The redundancy was 17.35%.Blast analysis showed that 39.5% of non-redundant sequences were homologous with the Nr database,14.81% with the EST database,and 32.1% were unknown sequences.Some sequences were related genes for flowering,insect and disease resistance.Therefore,the rubber tree methylation library is helpful for discovery and cloning of functional genes.