荷伯生氏斜盖伞的同核体菌株制备

【目的】笔者前期从栎树外生菌根根尖组织中分离得1株真菌:荷伯生氏斜盖伞(Clitopilus hobsonii),发现其对苗木具有促生效应。研究旨在获得该菌的同核体菌株,为荷伯生氏斜盖伞高质量基因组数据的获得及其对植物的促生作用机制研究奠定基础。【方法】通过原生质体再生技术得到候选菌株,基于候选菌株与出发菌株的生长性状及菌落形态特征比较、RNA聚合酶Ⅱ基因(RNA polymerase Ⅱ gene,rpb2)和翻译延伸因子基因(translation elongation factor 1-α gene,tef)两个单拷贝保守基因片段的杂合性分析,以及DAPI(4',6-二脒基-2-苯基吲哚)细胞核荧光染色观察,确定获得的再生菌株为同核体。【结果】经分离培养发现:其中一类再生菌株与出发菌株的菌落形态特性相比差异明显,且该再生菌株生长速度较慢;镜检结果表明,再生菌株与出发菌株均不存在锁状联合结构;DAPI细胞核荧光染色结果表明出发菌株菌丝细胞中均含有两个细胞核,而再生菌株初生菌丝中可观察到仅含1个细胞核的细胞结构,后期则发生双核化;rpb2和tef片段序列比对结果进一步表明出发菌株存在多个杂合位点,而在再生菌株中则未发现。【结论】本研究制备的再生菌株为荷伯生氏斜盖伞同核体菌株,具有单一、稳定的遗传信息,可为异核体真菌基因组数据的获得提供材料基础。

Homokaryotic strain obtained from Clitopilus hobsonii

【Objective】Our previous studies showed that the Clitopilus hobsonii recovered from ectomycorrhizal root tips in several Quercus species. This fungus was effective in promoting tree growth. The main purpose of this work is to generate the homokaryotic strains of this fungus for obtaining a high-quality genome assembly and better understanding its mechanism of promoting plant growth.【Method】This study was based on a protoplast regeneration technique to obtain the regenerated strain, comparing the colony morphology and growth rate differences between the regenerated and original strains using the RNA polymerase second largest subunit gene (rpb2) and nuclear translation elongation factor subunit 1-α (tef) as conservative genes. This was then performed on PCR identification, taking 4', 6-diamidino-2-phenylindole (DAPI) fluorescence nucleus staining together to determine if the regenerated strain is homokaryotic.【Result】We provided three lines of evidence to confirm that we had obtained the homokaryotic strain. First, we found that the colony appearance and the growth rate differed considerably between the regenerated and original strains, even though they both lack the clamp connection. Second, DAPI fluorescence nucleus staining and confocal microscopy confirmed that the heterokaryotic hypha contains two nuclei. In contrast, each hyphal cell has a single nucleus in the regenerated strain at its immature stage, while growing into homokaryotic hyphae that contain two nuclei at maturity. Third, sanger sequencing of rpb2 and tef partial genes revealed that the original strain showed a degree of heterozygosity, but this was completely absent in the regenerated strain. 【Conclusion】 Collectively, these data suggest that the regenerated strain of C. hobsonii is homokaryotic. With accurate and stable genetic information, we can provide a material basis for obtaining genomic data of heterokaryon fungi.