链霉菌看家基因引物的设计与验证

Design and validation of primers for housekeeping genes of streptomycetes

摘要:

看家基因的扩增与测序是进行多基因系统进化分析首先需要解决的问题。针对链霉菌这一群高(G+C)mol%革兰氏阳性细菌,选定4个看家基因:atpD、recA、rpoB和trpB,利用NCBI数据库中已有的2个链霉菌和3个分枝杆菌的全基因组序列,以及另两个链霉菌的recA基因序列,通过软件分析设计了各基因的扩增和测序引物,并优化了扩增反应条件。从所试验的55株链霉菌中,均特异地扩增出了上述4个基因的片段,并成功进行了序列测定,验证了所设计引物的实用性。所归纳的引物设计方法可用于高(G+C)mol%革兰氏阳性细菌的其它看家基因,以促进多基因系统进化研究的开展。

Abstract:

Multigene-based phylogenetic analyses are becoming more widely used in molecular taxonomy because of its lab-to-lab portability and reproducibility. The first step that needs to be settled for this approach is to amplify and sequence several housekeeping genes. Four housekeeping genes atpD, recA, rpoB and trpB were chosen for streptomycetes, which are representatives of high G+C mol% Gram-positive bacteria. Primer pairs for amplification and sequencing of the gene fragments were designed according to the known genome sequences of two streptomycetes and three mycobacteria, as well as the available recA gene sequences of another two streptomycetes in NCBI database, by using software packages Primer premier 5.0, Oligo 6.0 and SPCR 3.0, and NCBI BLAST program. Performance of the primer sets were validated by specific amplification of all gene fragments from the 55 streptomycete tested strains under optimized PCR reaction conditions, and by successful sequencing of the amplification products. It is concluded that the primer sets designed here are effective, and the primer design procedure and guidelines are valuable for other housekeeping genes of high G+C mol% bacteria.