Kreuzinger N, Podeu R, Gruber F, Göbl F, Kubicek C P
Abteilung für Mikrobielle Biochemie, Technische Universität Wien, Vienna, Austria.
Appl Environ Microbiol. 1996 Sep;62(9):3432-8. doi: 10.1128/aem.62.9.3432-3438.1996.
Degenerated oligonucleotide primers designed to flank an approximately 1.2-kb fragment of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd) from ascomycetes and basidiomycetes were used to amplify the corresponding gpd fragments from several species of the ectomycorrhizal fungal taxa Boletus, Amanita, and Lactarius. Those from B. edulis, A. muscaria, and L. deterrimus were cloned and sequenced. The respective nucleotide sequences of these gene fragments showed a moderate degree of similarity (72 to 76%) in the protein-encoding regions and only a low degree of similarity in the introns (56 to 66%). Introns, where present, occurred at conserved positions, but the respective positions and numbers of introns in a given taxon varied. The amplified fragment from a given taxon could be distinguished from that of others by both restriction nuclease cleavage analysis and Southern hybridization. A procedure for labeling DNA probes with fluorescein-12-dUTP by PCR was developed. These probes were used in a nonradioactive hybridization assay, with which the gene could be detected in 2 ng of chromosomal DNA of L. deterrimus on slot blots. Taxon-specific amplification was achieved by the design of specific oligonucleotide primers. The application of the gpd gene for the identification of mycorrhizal fungi under field conditions was demonstrated, with Picea abies (spruce) mycorrhizal roots harvested from a northern alpine forest area as well as from a plant-breeding nursery. The interference by inhibitory substances, which sometimes occurred in the DNA extracted from the root-fungus mixture, could be overcome by using very diluted concentrations of template DNA for a first round of PCR amplification followed by a second round with nested oligonucleotide primers. We conclude that gpd can be used to detect ectomycorrhizal fungi during symbiotic interaction.
设计了简并寡核苷酸引物，用于扩增子囊菌和担子菌中编码甘油醛-3-磷酸脱氢酶（gpd）的基因的一个约1.2 kb片段两侧的序列，并用其从外生菌根真菌类群牛肝菌属、鹅膏菌属和乳菇属的几个物种中扩增相应的gpd片段。对美味牛肝菌、毒蝇鹅膏菌和有害乳菇的gpd片段进行了克隆和测序。这些基因片段的相应核苷酸序列在蛋白质编码区显示出中等程度的相似性（72%至76%），而在内含子中仅显示出低度相似性（56%至66%）。内含子（如果存在）出现在保守位置，但给定分类群中内含子的各自位置和数量有所不同。通过限制性核酸酶切割分析和Southern杂交，可以将给定分类群的扩增片段与其他分类群的片段区分开来。开发了一种通过PCR用荧光素-12-dUTP标记DNA探针的方法。这些探针用于非放射性杂交分析，通过该分析可以在斑点印迹上2 ng的有害乳菇染色体DNA中检测到该基因。通过设计特异性寡核苷酸引物实现了分类群特异性扩增。证明了gpd基因在野外条件下用于鉴定菌根真菌的应用，使用了从北部高山森林地区以及植物育种苗圃采集的云杉菌根根。从根-真菌混合物中提取的DNA有时会出现抑制物质的干扰，可以通过使用非常稀释浓度的模板DNA进行第一轮PCR扩增，然后使用巢式寡核苷酸引物进行第二轮扩增来克服。我们得出结论，gpd可用于检测共生相互作用期间的外生菌根真菌。
