荷伯生氏斜盖伞的同核体菌株制备

doi:10.12302/j.issn.1000-2006.202108054

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南京林业大学学报（自然科学版） ›› 2022, Vol. 46 ›› Issue (5): 185-191.doi: 10.12302/j.issn.1000-2006.202108054

荷伯生氏斜盖伞的同核体菌株制备

王玉宸¹^,²^,³(), 王欣玉³, 彭龙¹^,³^,^*(), 袁志林¹^,³^,^*()

1.林木遗传育种国家重点实验室,中国林业科学研究院,北京 100091
2.南京林业大学林学院,江苏南京 210037
3.中国林业科学研究院亚热带林业研究所,浙江杭州 311400

收稿日期:2021-08-30 修回日期:2022-01-12 出版日期:2022-09-30 发布日期:2022-10-19
通讯作者: 彭龙,袁志林
基金资助:
国家自然科学基金青年基金项目(31901290);中央级公益性科研院所基本科研业务费专项资金项目(CAFYBB2020QB002)

Homokaryotic strain obtained from Clitopilus hobsonii

WANG Yuchen¹^,²^,³(), WANG Xinyu³, PENG Long¹^,³^,^*(), YUAN Zhilin¹^,³^,^*()

1. State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing 100091, China
2. College of Forestry, Nanjing Forestry University, Nanjing 210037, China
3. Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou 311400, China

Received:2021-08-30 Revised:2022-01-12 Online:2022-09-30 Published:2022-10-19
Contact: PENG Long,YUAN Zhilin

摘要/Abstract

摘要：

【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。

关键词: 担子菌, 原生质体, 同核体, 杂合度, 斜盖伞属

Abstract:

【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.

Key words: basidiomycetes, protoplast, homokaryotic, heterozygosity, Clitopilus

中图分类号:

S718.8

王玉宸,王欣玉,彭龙,等. 荷伯生氏斜盖伞的同核体菌株制备[J]. 南京林业大学学报（自然科学版）, 2022, 46(5): 185-191.

WANG Yuchen, WANG Xinyu, PENG Long, YUAN Zhilin. Homokaryotic strain obtained from Clitopilus hobsonii[J].Journal of Nanjing Forestry University (Natural Science Edition), 2022, 46(5): 185-191.DOI: 10.12302/j.issn.1000-2006.202108054.

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参考文献 36

[1]	JIAN S P, BAU T, ZHU X T, et al. Clitopilus, Clitella, and Clitopilopsis in China[J]. Mycologia, 2020, 112(2): 371-399. DOI: 10.1080/00275514.2019.1703089.
[2]	ORTON P D. New check list of British agarics and boleti[J]. Trans Br Mycol Soc, 1960, 43(2): 159-384. DOI: 10.1016/s0007-1536(60)80065-4.
[3]	PENG L, SHAN X L, YANG Y Z, et al. Potassium limitation promotes the Sweetgum-Clitopilus symbiosis[J]. Plant Cell Environ, 2021, DOI: 10.1111/pce.14053.
[4]	BALDRIAN P, KOHOUT P. Interactions of saprotrophic fungi with tree roots: can we observe the emergence of novel ectomycorrhizal fungi?[J]. New Phytol, 2017, 215(2): 511-513. DOI: 10.1111/nph.14665.
[5]	MARTIN F M, UROZ S, BARKER D G. Ancestral alliances: plant mutualistic symbioses with fungi and bacteria[J]. Science, 2017, 356(6340): eaad4501. DOI: 10.1126/science.aad4501.
[6]	CASADO LÓPEZ S, PENG M, DALY P, et al. Draft genome sequences of three monokaryotic isolates of the white-rot basidiomycete fungus Dichomitus squalens[J]. Microbiol Resour Announc, 2019, 8(18): e00264-19. DOI: 10.1128/mra.00264-19.
[7]	LU M Y, FAN W L, WANG W F, et al. Genomic and transcriptomic analyses of the medicinal fungus Antrodia cinnamomea for its metabolite biosynthesis and sexual development[J]. PNAS, 2014, 111(44): E4743-E4752. DOI: 10.1073/pnas.1417570111.
[8]	OHM R A, DE JONG J F, LUGONES L G, et al. Genome sequence of the model mushroom Schizophyllum commune[J]. Nat Biotechnol, 2010, 28(9): 957-963. DOI: 10.1038/nbt.1643.
[9]	潘迎捷, 陈明杰, 汪昭月, 等. 单核和同核原生质体技术在食用菌遗传育种上的应用[J]. 食用菌学报, 1994, 1(2): 56-62.
	PAN Y J, CHEN M J, WANG Z Y, et al. Application of monokaryon and homokaryon protoplast technology in genetic and breeding of edible mushroom[J]. Acta Edulis Fungi, 1994, 1(2): 56-62. DOI: 10.16488/j.cnki.1005-9873.1994.02.012.
[10]	ZHAO J, CHANG S T. Monokaryotization by protoplasting heterothallic species of edible mushrooms[J]. World J Microbiol Biotechnol, 1993, 9(5): 538-543. DOI: 10.1007/BF00386290.
[11]	CHEN S, XU J, LIU C, et al. Genome sequence of the model medicinal mushroom Ganoderma lucidum[J]. Nat Commun, 2012, 3: 913. DOI: 10.1038/ncomms1923.
[12]	NOGUEIRA-LÓPEZ G, PADILLA-ARIZMENDI F, INWOOD S, et al. TrichoGate: an improved vector system for a large scale of functional analysis of Trichoderma genes[J]. Front Microbiol, 2019, 10: 2794. DOI: 10.3389/fmicb.2019.02794.
[13]	ANDERSON J B, PETSCHE D M, HERR F B, et al. Breeding relationships among several species of Agaricus[J]. Can J Bot, 1984, 62(9): 1884-1889. DOI: 10.1139/b84-257.
[14]	CASTLE A J, HORGEN P A, ANDERSON J B. Crosses among homokaryons from commercial and wild-collected strains of the mushroom Agaricus brunnescens (=A. bisporus)[J]. Appl Environ Microbiol, 1988, 54(7): 1643-1648. DOI:10.1128/aem.54.7.1643-1648.1988.
[15]	SONNENBERG A S, WESSELS J G, GRIENSVEN L J. An efficient protoplasting/regeneration system for Agaricus bisporus and Agaricus bitorquis[J]. Curr Microbiol, 1988, 17(5): 285-291. DOI: 10.1007/BF01571330.
[16]	张妍, 黄晨阳, 高巍. 食用菌分子育种研究进展[J]. 菌物研究, 2019, 17(4): 229-239.
	ZHANG Y, HUANG C Y, GAO W. Research advances on molecular mushroom breeding[J]. J Fungal Res, 2019, 17(4): 229-239. DOI: 10.13341/j.jfr.2019.8015.
[17]	朱朝辉, 陈明杰, 谭琦, 等. 香菇原生质体单核体的再生与交配型的关系[J]. 食用菌学报, 2000, 7(4): 1-3.
	ZHU Z H, CHEN M J, TAN Q, et al. Study on the relationship between protoplasted monokaryon regeneration and mating types of Lentinula edodes[J]. Acta Edulis Fungi, 2000, 7(4): 1-3. DOI: 10.16488/j.cnki.1005-9873.2000.04.001.
[18]	JIN W, WANG Y C, SUN H J, et al. A survey of the potential ectomycorrhizal fungi associated with nursery seedlings of seven species of exotic Quercus in China[J]. J Sustain For, 2021, 40(4): 357-370. DOI: 10.1080/10549811.2020.1758151.
[19]	LIESCHE J, ZIOMKIEWICZ I, SCHULZ A. Super-resolution imaging with Pontamine fast scarlet 4BS enables direct visualization of cellulose orientation and cell connection architecture in onion epidermis cells[J]. BMC Plant Biol, 2013, 13: 226. DOI: 10.1186/1471-2229-13-226.
[20]	鲍大鹏, 杨瑞恒, 王莹, 等. 运用萎锈灵抗性基因构建香菇聚乙二醇介导的遗传转化方法[J]. 微生物学通报, 2018, 45(8): 1824-1828.
	BAO D P, YANG R H, WANG Y, et al. PEG-mediated genetic transformation of Lentinula edodes by using carboxin resistant gene as selective marker[J]. Microbiol China, 2018, 45(8): 1824-1828. DOI: 10.13344/j.microbiol.china.170861.
[21]	胡晓棣, 李熠, 任蜀豫, 等. 冬虫夏草、蛹虫草菌丝隔膜和细胞核荧光染色[J]. 菌物学报, 2016, 35(9): 1099-1105.
	HU X D, LI Y, REN S Y, et al. Fluorescent staining of septa and nuclei in Ophiocordyceps sinensis and Cordyceps militaris[J]. Mycosystema, 2016, 35(9): 1099-1105. DOI: 10.13346/j.mycosystema.150139.
[22]	LIU Y J, WHELEN S, HALL B D. Phylogenetic relationships among ascomycetes: evidence from an RNA polymerse II subunit[J]. Mol Biol Evol, 1999, 16(12): 1799-1808. DOI: 10.1093/oxfordjournals.molbev.a026092.
[23]	REHNER S A, BUCKLEY E. A Beauveria phylogeny inferred from nuclear ITS and EF1-alpha sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs[J]. Mycologia, 2005, 97(1): 84-98. DOI: 10.3852/mycologia.97.1.84.
[24]	丛倩倩, 崔晓, 王庆武. 灵芝原生质体单核菌株的制备及形态特性比较[J]. 食用菌, 2019, 41(2): 34-36.
	CONG Q Q, CUI X, WANG Q W. Preparation of protoplast monokaryon strains and comparison of morphological characteristics of Gannoderma ludidum[J]. Edible Fungi, 2019, 41(2): 34-36.
[25]	徐思佳, 万佳宁, 李焱, 等. 刺芹侧耳交配型基因敲入单核体后锁状联合和核相的表征观察[J]. 菌物学报, 2020, 39(6): 1130-1138.
	XU S J, WAN J N, LI Y, et al. Characterization of clamps and nuclear phases in the monokaryons with transformed mating genes in Pleurotus eryngii[J]. Mycosystema, 2020, 39(6): 1130-1138. DOI: 10.13346/j.mycosystema.200085.
[26]	HUANG C Y, XU J Y, GAO W, et al. A reason for overlap peaks in direct sequencing of rRNA gene ITS in Pleurotus nebrodensis[J]. FEMS Microbiol Lett, 2010, 305(1): 14-17. DOI: 10.1111/j.1574-6968.2009.01891.x.
[27]	SINGER R. The Agaricales in modern taxonomy[M]// Koeltz Scientific Books. 4th ed. Germany: Koenigstein, 1986: 699-700.
[28]	HANSEN E M. Nuclear condition and vegetative characteristics of homokaryotic and heterokaryotic isolates of Phellinus weirü[J]. Can J Bot, 1979, 57(15): 1579-1582. DOI: 10.1139/b79-196.
[29]	GAO Q, YAN D, WANG D, et al. Variations in nuclear number and size in vegetative hyphae of the edible mushroom Lentinula edodes[J]. Front Microbiol, 2019, 10: 1987. DOI: 10.3389/fmicb.2019.01987.
[30]	华丰. 离子色谱法测定灵芝超声水提取物中4种代表有机酸的含量[J]. 生物加工过程, 2021, 19(1):74-78.
	HUA F. Determination of four organic acids in Ganoderma lucidum ultrasonic water extract by ion chromatography[J]. Chi J Bio Eng, 2021, 19(1):74-78.DOI:10.3969/j.issn.1672-3678.2021.01.011.
[31]	HA B, LEE S, KIM S, et al. Nucleus-selective expression of laccase genes in the dikaryotic strain of Lentinula edodes[J]. Mycobiology, 2017, 45(4): 379-384. DOI: 10.5941/MYCO.2017.45.4.379.
[32]	LARRAYA L M, IDARETA E, ARANA D, et al. Quantitative trait loci controlling vegetative growth rate in the edible basidiomycete Pleurotus ostreatus[J]. Appl Environ Microbiol, 2002, 68(3): 1109-1114. DOI: 10.1128/aem.68.3.1109-1114.2002.
[33]	RAUDASKOSKI M. The central role of septa in the basidiomycete Schizophyllum commune hyphal morphogenesis[J]. Fungal Biol, 2019, 123(9): 638-649. DOI: 10.1016/j.funbio.2019.05.009.
[34]	雷晓娥, 乔燕楠, 毛景秀, 等. 基于转录组分析香菇不同漆酶活性单核菌丝体基因表达[J]. 菌物学报, 2020, 39(6): 1162-1174.
	LEI X E, QIAO Y N, MAO J X, et al. Comparative analyses of gene expression based on transcriptome data in different laccase activities of monokaryotic mycelia of Lentinula edodes[J]. Mycosystema, 2020, 39(6): 1162-1174. DOI: 10.13346/j.mycosystema.200091.
[35]	KIM S, SONG Y, HA B, et al. Variable number tandem repeats in the mitochondrial DNA of Lentinula edodes[J]. Genes, 2019, 10(7): 542. DOI: 10.3390/genes10070542.
[36]	YE L Y, DENG Y J, MUKHTAR I, et al. Mitochondrial genome and diverse inheritance patterns in Pleurotus pulmonarius[J]. J Microbiol, 2020, 58(2): 142-152. DOI:10.1007/s12275-020-9318-x.

菌株材料 strains type	菌落生长速度/ (mm·d^-1) mycelium growth rate	菌丝致密程度 mycelium density
再生菌株regenerated strains	2.82±0.03 b	+++
出发菌株original strains	5.72±0.07 a	+

荷伯生氏斜盖伞的同核体菌株制备

Homokaryotic strain obtained from Clitopilus hobsonii

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