Discovery and Verification of a Functional Gene Influencing the Growth and Development of Pleurotus ostreatus

doi:10.13560/j.cnki.biotech.bull.1985.2024-1257

Abstract

Abstract:

Objective This study is aimed to investigate the role of the functionally unknown gene g13394 in the growth and development of Pleurotus ostreatus, specifically in the transition from mycelium to primordium and fruiting body formation. Method The g13394 gene was cloned from the P. ostreatus strain CCMSSC00389 using Polymerase Chain Reaction (PCR), and its biological characteristics were analyzed through bioinformatic tools. A homologous recombination approach was used to construct both overexpression and RNA interference (RNAi) vectors, which were then introduced into P. ostreatus via Agrobacterium tumefaciens-mediated transformation. Gene expressions in the transformed strains were validated by quantitative real-time PCR (qRT-PCR) to select the g13394-overexpressed and RNAi strains. The phenotypic differences between the overexpression, RNAi, and wild-type strains were assessed at the mycelial growth stage and during fruiting body development. Result Bioinformatic analysis revealed that g13394 encoded a nuclear-localized protein with unknown functional domains and the subcellular localization was predicted in the nucleus. Two strains were successfully constructed and obtained for g13394 overexpression and RNAi, respectively. Overexpressed strains showed significantly faster mycelial growth and accelerated fruiting body development compared to the wild-type. In contrast, RNAi strains showed slower mycelial growth, delayed primordium formation, and extended fruiting body development time. These results suggest that g13394 positively regulates both mycelial growth and the timing of fruiting body formation. Conclusion The gene g13394 promotes mycelial growth and accelerates the formation of fruiting bodies.

Key words: Pleurotus ostreatus, gene cloning, genetic transformation, fruiting body development

PEI Jing-qi, ZHAO Meng-ran, HUANG Chen-yang, WU Xiang-li. Discovery and Verification of a Functional Gene Influencing the Growth and Development of Pleurotus ostreatus[J]. Biotechnology Bulletin, 2025, 41(6): 327-334.

Figures/Tables 7

Table 1 List of primers

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	用途 Purpose
g13394-F	ATGAACGAAGTCTCGGG	克隆
g13394-R	TCACGCGCCCTCTGCATTTA
OE13-F	ACTGACCTGGGGATCCATGAACGAAGTCTCGGGGCG
OE13-R	GCATGCCAATTCTAGATCACGCGCCCTCTGCATTTAACACG
Ri-sense-g13394 F	ACTGACCTGGGGATCCTCACGCGCCCTCTGCATT
Ri-sense-g13394 R	GCATGCCAATTCTAGAACGAAAGAGAGAGCATCCAAGGC
Ri-anti-g13394 F	TCTCTTTCGTTCTAGACGGATGTCCTCTACGAGGGA
Ri-anti-g13394 R	GGCCAGTGCCAAGCTTTCACGCGCCCTCTGCATT
q13-F	CGCTCACCTGCTCAATACG	RT-qPCR引物
q13-R	TCCATGACCCTGCCAATTG	RT-qPCR引物
tub-F	AGGCTTTCTTGCATTGGTACACGC	内参引物
tub-R	TATTCGCCTTCTTCCTCATCGGCA	内参引物
S10-F	CCACACAAATCCTCTCACTCACG	验证引物
S10-R	GCTTGCATGCCTGCAGTAATT
Hyg-F	CGACAGATCCGGTCGGCATCTACTCTATTTCTT
Hyg-R	TCTCGTGCTTTCAGCTTCGATGTAGGAGGG

Fig. 1 Bioinformatics analysis of g13394A: Gene structure; B: transmembrane structural domain analysis; C: signal peptide prediction; D: phosphorylation site prediction; E: protein structural domain prediction

Fig. 2 PCR amplification of g13394 DNA and CDS

Fig. 3 g13394 overexpression and RNAi plasmid profilesA: Plasmid Po-gpdOE-g13394; B: plasmid RNAi-g13394

Fig. 4 PCR verification of g13394 putative transformants and the relative expressionA: Overexpression positive transformants; B: RNAi positive transformants; M: DL2000 marker; 1-20: g13394 transformants; CK: positive control (g13394 overexpression/RNAi plasmid); NC1: negative control (WT); NC2: negative control (ddH2O); C: Overexpressing positive transformants; D: RNAi transformants; WT: wild type; OE-2, OE-7, OE-9, OE-10, and OE-13 correspond to lane 1, 6, 8, 9, 12 in Fig. 4-A. Ri-12, Ri-13, Ri-23, Ri-32, and Ri-34 correspond to lane 1, 2, 7, 16, 18 in Fig. 4-B. Diﬀerent letters indicate signiﬁcant diﬀerences among strains at P<0.05. The same below

Fig. 5 Mycelial growth of wild-type strain and g13394 transformed strain on PDA mediumA: Colony morphology. B: Mycelial growth rate

Fig. 6 Fruiting body development of the wild-type strain and g13394 transformantsA: Fruiting body morphology; M: mycelium (12 d); P: primordium (25 d); F: fruiting body (29 d). B: Primordium formation time. C: Fruiting body formation time

References 32

1	Liu Q, Kong WL, Cui X, et al. Dynamic succession of microbial compost communities and functions during Pleurotus ostreatus mushroom cropping on a short composting substrate [J]. Front Microbiol, 2022, 13: 946777.
2	Li X, Liu MQ, Dong CH. Hydrophobin gene Cmhyd4 negatively regulates fruiting body development in edible fungi Cordyceps militaris [J]. Int J Mol Sci, 2023, 24(5): 4586.
3	Lyu XM, Jiang SY, Wang L, et al. The Fvclp1 gene regulates mycelial growth and fruiting body development in edible mushroom Flammulina velutipes [J]. Arch Microbiol, 2021, 203(9): 5373-5380.
4	Lyu XM, Wang QJ, Liu A, et al. The transcription factor Ste12-like increases the mycelial abiotic stress tolerance and regulates the fruiting body development of Flammulina filiformis [J]. Front Microbiol, 2023, 14: 1139679.
5	Tao YX, Chen RL, Yan JJ, et al. A hydrophobin gene, Hyd9, plays an important role in the formation of aerial hyphae and primordia in Flammulina filiformis [J]. Gene, 2019, 706: 84-90.
6	Li ZH, Zhou YY, Xu CT, et al. Genome-wide analysis of the Pleurotus eryngii laccase gene (PeLac) family and functional identification of PeLac5 [J]. AMB Express, 2023, 13(1): 104.
7	Liu QZ, Qu S, He GQ, et al. Mating-type genes play an important role in fruiting body development in Morchella sextelata [J]. J Fungi, 2022, 8(6): 564.
8	Hou LD, Liu ZQ, Yan KX, et al. Mnsod1 promotes the development of Pleurotus ostreatus and enhances the tolerance of mycelia to heat stress [J]. Microb Cell Fact, 2022, 21(1): 155.
9	Hou LD, Wang LN, Wu XL, et al. Expression patterns of two pal genes of Pleurotus ostreatus across developmental stages and under heat stress [J]. BMC Microbiol, 2019, 19(1): 231.
10	王丽宁. 糙皮侧耳过氧化氢酶基因特征分析和功能研究 [D]. 北京: 中国农业科学院, 2019.
	Wang LN. Characteristics and function of catalase gene in Pleurotus ostreatus [D]. Beijing: Chinese Academy of Agricultural Sciences, 2019.
11	Pei JQ, Zhao MR, Zhang LJ, et al. The metacaspase gene PoMCA1 enhances the mycelial heat stress tolerance and regulates the fruiting body development of Pleurotus ostreatus [J]. Horticulturae, 2024, 10(2): 116.
12	Song LH, Ciftci-Yilmaz S, Harper J, et al. Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants expressing proteins of unknown function [J]. Plant Physiol, 2008, 148(1): 280-292.
13	Ricoux R, Boucher JL, Mansuy D, et al. Microperoxidase 8 catalyzed nitration of phenol by nitrogen dioxide radicals [J]. Eur J Biochem, 2001, 268(13): 3783-3788.
14	Lei M, Wu XL, Zhang JX, et al. Establishment of an efficient transformation system for Pleurotus ostreatus [J]. World J Microbiol Biotechnol, 2017, 33(12): 214.
15	侯志浩. 糙皮侧耳Zn₂Cys₆转录因子家族的表达分析及PoZCP26的功能研究 [D]. 北京: 中国农业科学院, 2020.
	Hou ZH. Expression analysis of Zn₂Cys₆ transcription factor family in Pleurotus ostreatus and functional study of PoZCP26 [D]. Beijing: Chinese Academy of Agricultural Sciences, 2020.
16	Chen SC, Ge W, Buswell JA. Molecular cloning of a new laccase from the edible straw mushroom Volvariella volvacea: possible involvement in fruit body development [J]. FEMS Microbiol Lett, 2004, 230(2): 171-176.
17	边银丙. 食用菌栽培学 [M]. 3版. 北京: 高等教育出版社, 2017.
	Bian YB. Edible mushroom cultivation [M]. 3rd ed. Beijing: Higher Education Press, 2017.
18	de Jong JF, Ohm RA, de Bekker C, et al. Inactivation of Ku80 in the mushroom-forming fungus Schizophyllum commune increases the relative incidence of homologous recombination [J]. FEMS Microbiol Lett, 2010, 310(1): 91-95.
19	Peñas MM, Asgeirsdóttir SA, Lasa I, et al. Identification, characterization, and in situ detection of a fruit-body-specific hydrophobin of Pleurotus ostreatus [J]. Appl Environ Microbiol, 1998, 64(10): 4028-4034.
20	Qi YC, Chen HJ, Zhang MK, et al. Identification and expression analysis of Pofst3 suggests a role during Pleurotus ostreatus primordia formation [J]. Fungal Biol, 2019, 123(3): 200-208.
21	Gollery M, Harper J, Cushman J, et al. What makes species unique? The contribution of proteins with obscure features [J]. Genome Biol, 2006, 7(7): R57.
22	Gollery M, Harper J, Cushman J, et al. POFs: what we don't know can hurt us [J]. Trends Plant Sci, 2007, 12(11): 492-496.
23	Zhang Y, Zhang F, Huang XZ. Characterization of an Arabidopsis thaliana DUF761-containing protein with a potential role in development and defense responses [J]. Theor Exp Plant Physiol, 2019, 31(2): 303-316.
24	Zhou XY, Zhu XG, Shao WN, et al. Genome-wide mining of wheat DUF966 gene family provides new insights into salt stress responses [J]. Front Plant Sci, 2020, 11: 569838.
25	Chen K, Wang YL, Nong XY, et al. Characterization and in silico analysis of the domain unknown function DUF568-containing gene family in rice (Oryza sativa L.) [J]. BMC Genomics, 2023, 24(1): 544.
26	Sánchez-Castro I, Gómez C, García J, et al. Role of certain DUF proteins in Agaricus bisporus development and environmental stress responses [J]. Fungal Biology Reviews, 2019, 32(3): 143-150.
27	López-García M, Pérez A, Hernández F, et al. Functional analysis of a DUF protein in white rot fungus reveals its role in mycelial growth and fruiting body formation [J]. Fungal Biology, 2020, 124(2): 120-129.
28	Rathore M, Kumar V, Singh S, et al. Multiple unknown function genes in Aspergillus niger are involved in spore formation and growth regulation [J]. Fungal Genetics and Biology, 2018, 112(1): 87-95.
29	Zhang L, Li J, Wang S, et al. Functional analysis of a novel DUF protein in Pleurotus ostreatus reveals its role in fruiting body development [J]. Fungal Genetics and Biology, 2020, 138(1): 103-112.
30	Li Y, Chen W, Zhou M, et al. Identification and characterization of a DUF protein involved in mycelial growth and fruiting body formation in Lentinula edodes [J]. Mycologia, 2019, 111(6): 123-131.
31	Wang H, Zhao X, Zhang Y, et al. A novel DUF protein regulates sexual development in Schizophyllum commune [J]. Fungal Biology, 2018, 122(2): 99-106.
32	Chen G, Lin Y, Xu Z, et al. Functional characterization of a DUF protein in Coprinopsis cinerea reveals its involvement in fruiting body development [J]. Fungal Genetics and Biology, 2017, 96(1): 73-80.

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