Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, College of Life Sciences, China Jiliang University, Hangzhou, 310018, China.
Curr Microbiol. 2013 Sep;67(3):263-70. doi: 10.1007/s00284-013-0356-z. Epub 2013 Apr 3.
Yeast-like symbiotes (YLS) are endosymbionts that are intimately associated with the growth, development, reproduction of their host, the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae). However, it is unclear how many species of YLS are found within N. lugens, and how they are related to each other. Traditional methods or simple amplification based on 18S rDNA sequence does not reliably identify new species quickly and efficiently. Therefore, a novel nested PCR-denaturing gradient gel electrophoresis (DGGE) strategy was developed in this article to analyze the YLS of brown planthopper using a nested PCR protocol that involved the 18S rDNA gene and the 5.8S-ITS gene using fungal universal primers. The nested PCR protocol was developed as follows: firstly, the 18S rDNA gene, and 5.8S-ITS gene were amplified using fungal universal primers. Subsequently, these products were used as a template in a second PCR with primers ITS1GC-ITS2, ITS1FGC-ITS2, and NFGC-NR, which was suitable for DGGE. Using this highly specific molecular approach, we found several previously detected fungi: Noda, Pichia guilliermondii, Candida sp., and some previously undetected fungi, such as Saccharomycetales sp., Debaryomyces hansenii, and some uncultured fungi. In conclusion, the nested PCR system developed in this study, coupled with DGGE fingerprinting, offers a new tool for uncovering fungal endosymbiont diversity within planthoppers.
酵母样共生体 (YLS) 是与它们的宿主褐飞虱（半翅目：飞虱科）的生长、发育和繁殖密切相关的内共生体。然而，目前尚不清楚褐飞虱体内有多少种 YLS，以及它们之间的关系如何。传统的方法或基于 18S rDNA 序列的简单扩增并不能快速有效地可靠识别新物种。因此，本文开发了一种新型嵌套 PCR-变性梯度凝胶电泳（DGGE）策略，使用涉及 18S rDNA 基因和 5.8S-ITS 基因的嵌套 PCR 方案，使用真菌通用引物来分析褐飞虱的 YLS。嵌套 PCR 方案如下：首先，使用真菌通用引物扩增 18S rDNA 基因和 5.8S-ITS 基因。随后，将这些产物用作模板，进行第二轮 PCR，使用引物 ITS1GC-ITS2、ITS1FGC-ITS2 和 NFGC-NR，这适合 DGGE。使用这种高度特异性的分子方法，我们发现了一些以前检测到的真菌：Noda、毕赤酵母、假丝酵母属和一些以前未检测到的真菌，如 Saccharomycetales sp.、德巴利酵母和一些未培养的真菌。总之，本研究中开发的嵌套 PCR 系统与 DGGE 指纹图谱相结合，为揭示飞虱体内真菌共生体的多样性提供了一种新工具。
