Paulsrud P, Lindblad P
Department of Physiological Botany, Uppsala University, Sweden.
Appl Environ Microbiol. 1998 Jan;64(1):310-5. doi: 10.1128/AEM.64.1.310-315.1998.
We examined the genetic diversity of Nostoc symbionts in some lichens by using the tRNA(Leu) (UAA) intron as a genetic marker. The nucleotide sequence was analyzed in the context of the secondary structure of the transcribed intron. Cyanobacterial tRNA(Leu) (UAA) introns were specifically amplified from freshly collected lichen samples without previous DNA extraction. The lichen species used in the present study were Nephroma arcticum, Peltigera aphthosa, P. membranacea, and P. canina. Introns with different sizes around 300 bp were consistently obtained. Multiple clones from single PCRs were screened by using their single-stranded conformational polymorphism pattern, and the nucleotide sequence was determined. No evidence for sample heterogenity was found. This implies that the symbiont in situ is not a diverse community of cyanobionts but, rather, one Nostoc strain. Furthermore, each lichen thallus contained only one intron type, indicating that each thallus is colonized only once or that there is a high degree of specificity. The same cyanobacterial intron sequence was also found in samples of one lichen species from different localities. In a phylogenetic analysis, the cyanobacterial lichen sequences grouped together with the sequences from two free-living Nostoc strains. The size differences in the intron were due to insertions and deletions in highly variable regions. The sequence data were used in discussions concerning specificity and biology of the lichen symbiosis. It is concluded that the tRNA(Leu) (UAA) intron can be of great value when examining cyanobacterial diversity.
我们通过使用tRNA(Leu)(UAA)内含子作为遗传标记，研究了一些地衣中念珠藻共生体的遗传多样性。在转录内含子二级结构的背景下分析核苷酸序列。无需事先提取DNA，就能从新鲜采集的地衣样本中特异性扩增出蓝细菌tRNA(Leu)(UAA)内含子。本研究中使用的地衣物种有北极肾衣、口疮皮果衣、膜质皮果衣和犬皮果衣。始终能获得大小约为300 bp的不同内含子。通过单链构象多态性模式筛选单个PCR产生的多个克隆，并测定核苷酸序列。未发现样本异质性的证据。这意味着原位共生体不是蓝藻共生体的多样化群落，而是单一的念珠藻菌株。此外，每个地衣叶状体仅包含一种内含子类型，这表明每个叶状体仅被定殖一次，或者存在高度特异性。在来自不同地点的一种地衣物种的样本中也发现了相同的蓝细菌内含子序列。在系统发育分析中，蓝细菌地衣序列与来自两个自由生活的念珠藻菌株的序列聚在一起。内含子的大小差异是由于高变区的插入和缺失造成的。序列数据被用于有关地衣共生特异性和生物学的讨论。得出的结论是，tRNA(Leu)(UAA)内含子在研究蓝细菌多样性时可能具有很大价值。
