难培养菌的多条带PCR产物产生原因分析

Analysis on causes of polymorphic PCR bands in nonculturable endophytes amplification

摘要:

【目的】分析严重威胁柑橘产业发展的毁灭性病害——柑橘黄龙病的强致病性病原亚洲种“Candidatus Liberibacter asiaticus”的种群多样性研究中出现多条带PCR产物的原因，为难培养菌的分子生物学研究提供参考。【方法】通过使用PCR、聚丙烯酰胺凝胶电泳(PAGE)和测序相结合分析“Ca. L. asiaticus”基因组上2个短串联重复(Short tandem repeats，STR)基因的PCR产物。【结果】单个菌株可扩增获得多个PCR条带且其扩增产物多态性受寄主品种影响；这2个STR基因的PCR产物在菌株间呈明显的多态性；扩增所获得的序列可来自“Ca. L. asiaticus”本身，也可来自其寄主或内生菌。【结论】难培养菌的STR基因PCR产物多态性产生的主要原因是该基因内部的串联重复序列数目存在差异，但目的菌及外源物种(寄主或内生菌等)基因组的非特异性扩增也是影响其多态性的因素。

Abstract:

[Objective] This study was to investigate the genetic nature of multiple amplicons from PCR experiments on ‘Candidatus Liberibacter asiaticus’ infected citrus samples and to provide reference for molecular biological studies of fastidious prokaryotes. [Methods] Two short tandem repeats (STR) loci in the ‘Ca. L. asiaticus’ genome were amplified by conventional PCR. PCR products were separated by PAGE. Polymorphic bands were collected, cloned, and sequenced. [Results] The primers from the two STR loci generated multiple amplicons from a single ‘Ca. L. asiaticus’ infected citrus sample. Sequence analyses showed that these amplicons could be from ‘Ca. L. asiaticus’, other bacteria and citrus host. [Conclusion] The main source of amplicon variations was due to changes of STR numbers in the targeted loci. However, amplification of host and endophytic bacterial DNA could also contribute to the multi-band phenomenon.