Development of LNA oligonucleotide–PCR clamping technique in investigating the community structures of plant-associated bacteria

Simultaneous extraction of plant organelle (mitochondria and plastid) genes during the DNA extraction step is major limitation in investigating the community structures of plant-associated bacteria. Although locked nucleic acid (LNA) oligonucleotides was designed to selectively amplify the bacterial small subunit rRNA genes by applying the PCR clamping technique, those for plastids were applicable only for particular plants, while those for mitochondria were available throughout most plants. To widen the applicable range, new LNA oligonucleotides specific for plastids were designed, and the efficacy was investigated. PCR without LNA oligonucleotides predominantly amplified the organelle genes, while bacterial genes were predominantly observed in having applied the LNA oligonucleotides. Denaturing gradient gel electrophoresis (DGGE) analysis displayed additional bacterial DGGE bands, the amplicons of which were prepared using the LNA oligonucleotides. Thus, new designed LNA oligonucleotides specific for plastids were effective and have widened the scope in investigating the community structures of plant-associated bacteria.

在DNA提取步骤中同时获取植物细胞器（线粒体与质体）基因，是探究植物相关细菌群落结构的主要限制因素。尽管锁核酸（locked nucleic acid, LNA）寡核苷酸可通过PCR钳制技术选择性扩增细菌小亚基rRNA基因，但针对质体的LNA寡核苷酸仅适用于特定植物，而针对线粒体的LNA寡核苷酸则可在多数植物中使用。为扩大适用范围，本研究设计了新型特异性靶向质体的LNA寡核苷酸，并对其扩增效果进行了验证。未添加LNA寡核苷酸的PCR反应主要扩增细胞器基因，而添加LNA寡核苷酸的反应则主要扩增细菌基因。变性梯度凝胶电泳（Denaturing gradient gel electrophoresis, DGGE）分析显示，使用该LNA寡核苷酸制备的扩增产物可检测到更多的细菌DGGE条带。综上，本次设计的新型靶向质体的LNA寡核苷酸具备良好的特异性，有效拓宽了植物相关细菌群落结构研究的适用范围。