氧酰基载体蛋白还原酶基因FgOAR1对禾谷镰孢生长、发育和致病的影响

doi:10.3864/j.issn.0578-1752.2023.24.005

摘要/Abstract

摘要：

【背景】禾谷镰孢（Fusarium graminearum）是引起小麦赤霉病（Fusarium head blight）的主要病原真菌，侵染后导致作物品质和产量下降，同时产生多种真菌毒素，严重危害粮食生产和人类健康。氧酰基载体蛋白还原酶（3-oxoacyl ACP reductase，OAR1）催化羧酰基载体蛋白还原成氧酰基载体蛋白，在脂肪酸的生物合成过程中具有重要作用。【目的】构建FgOAR1基因缺失突变体，研究其表型、显微结构、孢子产量、有性生殖和致病力等的变化，探究FgOAR1的生物学功能，揭示其对禾谷镰孢生长、发育和致病的影响，为新型抑菌作用靶点的筛选及小麦赤霉病的防控提供科学依据。【方法】以禾谷镰孢野生型菌株PH-1为材料，应用Split-Marker基因敲除技术，构建FgOAR1基因缺失突变体（ΔFgOAR1），Gap-repair技术获得缺失基因的回补突变体（ΔFgOAR1-C）；将PH-1、ΔFgOAR1和ΔFgOAR1-C菌株分别接种到常规培养基，观察菌落表型变化并测定脂肪酸含量差异；接种到含刚果红（Congo-Red）、SDS、NaCl和H₂O₂的压力筛选培养基中，观察菌丝在压力培养条件下的生长状况；接种CMC培养基，比较分生孢子产量差异；接种胡萝卜培养基，观察有性生殖及孢子性状；分别进行小麦胚芽鞘和穗部接种，统计发病率和毒素产量，比较突变体与野生型菌株的致病力差异。【结果】与野生型PH-1菌株相比，在PDA、YEG和CM培养基中ΔFgOAR1突变体未呈现明显表型变化，在0.7 mol·L^-1 NaCl、0.03% H₂O₂、0.01% SDS和300 μg·mL^-1 Congo-Red等压力培养条件下，突变体菌落直径显著低于野生型PH-1，表明FgOAR1与禾谷镰孢细胞膜和细胞壁的合成相关；ΔFgOAR1突变体在液体培养基中的分生孢子产量（8.1×10⁵个/mL）显著低于PH-1（1.26×10⁶个/mL），接种小麦胚芽鞘和麦穗后，ΔFgOAR1突变体的致病力显著低于野生型菌株PH-1。【结论】禾谷镰孢中FgOAR1参与脂肪酸的生物合成，对细胞膜合成具有重要作用，该基因缺失导致突变体抗逆性降低，分生孢子产量下降，且致病力显著降低，FgOAR1对禾谷镰孢的生长、发育和致病具有重要作用。

关键词: 禾谷镰孢, 小麦赤霉病, 基因敲除, FgOAR1, 致病力

Abstract:

【Background】Fusarium graminearum is the main pathogenic fungus which can infect wheat and result in Fusarium head blight. The infection of F. graminearum leads to huge reduction in crop quality and yield, and produces different types of mycotoxins that endanger grain production and human health. 3-oxoacyl ACP reductase (OAR1), catalyzing carboxyacyl carrier protein to oxyacyl carrier protein, which plays an important role in the fatty acid biosynthesis of F. graminearum.【Objective】To reveal the biological function of FgOAR1 in F. graminearum, the FgOAR1 deletion mutants were constructed. The phenotype, cell structure, conidia concentration, sexual reproduction and pathogenicity of mutant were analyzed and compared with wild-type strain PH-1. The results will uncover the function of FgOAR1 in the growth and pathogenic process of F. graminearum, and provide scientific evidences for the identification of novel antifungal target and control of Fusarium head blight.【Method】Wild-type strain F. graminearum PH-1 was used as material in this study. Split-Marker gene knockout technology was conducted to construct the ΔFgOAR1 mutants. The ΔFgOAR1-C reverting strain was obtained by the Gap-repair method. All strains were individually inoculated in common media (PDA, CM, YPG), and the stress selection medium containing Congo-Red, SDS, NaCl and H₂O₂, respectively. The mycelium phenotypes and fatty acid content were recorded. The strains were inoculated in CMC medium to analyze the conidia concentration. The carrot culture-medium was used to analyze the sexual reproduction of each strain. All strains were individually inoculated to the wheat coleoptile and spikelet to analyze the pathogenicity and mycotoxin content, respectively. The disease incidence of mutant strains was calculated and compared with PH-1 strain.【Result】Compared with the wild-type strain PH-1, the growth of ΔFgOAR1 mutant showed no difference in PDA, YEG and CM media. Whereas, under the pressure of 0.7 mol·L^-1 NaCl, 0.03% H₂O₂, 0.01% SDS and 300 μg·mL^-1 Congo-Red, the growth of ΔFgOAR1 mutant was significantly reduced compared with PH-1. The evidence demonstrated that FgOAR1 is related to the cell membrane and cell wall formation in F. graminearum. In CMC medium, the conidia concentration of ΔFgOAR1 mutant (8.1×10⁵ conidia/mL) was less than PH-1 (1.26×10⁶ conidia/mL) with significant difference; The pathogenicity analysis further demonstrated that the disease incidence of ΔFgOAR1 mutant was significantly lower than PH-1 in wheat coleoptile and spikelet inoculation tests.【Conclusion】FgOAR1 participates in the fatty acid biosynthesis, and plays an important role in cell membrane formation of F. graminearum. The deletion of FgOAR1 resulted in the reduced resistance to pressures, decreased conidia production and pathogenicity. Therefore, FgOAR1 is important for the growth, development and pathogenicity process of F. graminearum.

Key words: Fusarium graminearum, Fusarium head blight, gene knockout, FgOAR1, pathogenicity

宫安东, 雷银玉, 吴楠楠, 刘景榕, 宋梦鸽, 张艺美, 杨光, 杨鹏. 氧酰基载体蛋白还原酶基因FgOAR1对禾谷镰孢生长、发育和致病的影响[J]. 中国农业科学, 2023, 56(24): 4854-4865.

GONG AnDong, LEI YinYu, WU NanNan, LIU JingRong, SONG MengGe, ZHANG YiMei, YANG Guang, YANG Peng. The Effect of 3-Oxyacyl ACP Reductase Gene FgOAR1 on the Growth, Development and Pathogenicity of Fusarium graminearum[J]. Scientia Agricultura Sinica, 2023, 56(24): 4854-4865.

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引物名称<BOLD>P</BOLD>rimer name	引物序列<BOLD>P</BOLD>rimer sequence (5′→3′)
FgOAR1_1F	TCTCGATACCTCCTACCTACCT
FgOAR1_2R	TTGACCTCCACTAGCTCCAGCCAAGCCTCAACACATGCTCTACACCCT
FgOAR1_3F	GAATAGAGTAGATGCCGACCGCGGGTTCCAGGGTCTCAAAGCAAC
FgOAR1_4R	ATCCCAGAAAATCCGCAA
HYG/F	GGCTTGGCTGGAGCTAGTGGAGGTCAA
HY/R	GTATTGACCGATTCCTTGCGGTCCGAA
YG/F	GATGTAGGAGGGCGTGGATATGTCCT
HYG/R	AACCCGCGGTCGGCATCTACTCTATTC
FgOAR1_5F	ATCGGAGTTGAGGTGACAG
FgOAR1_6R	GAAGAGGGGAGGAAAGGTT
FgOAR1_7F	GGTCAATAGAGCAGGTCGC
FgOAR1_8R	CAGGCGGTGGTAACTTCA
h850	TTCCTCCCTTTATTTCAGATTCAA
h852	ATGTTGGCGACCTCGTATTGG
h855R	GTCGATGCGACGCAATCGT
h856F	GCTGATCTGACCAGTTGC
FgOAR1_hbF	CGACTCACTATAGGGCGAATTGGGTACTCAAATTGGTGACAGCGGCGTTAGGACA
FgOAR1_hbR	CACCACCCCGGTGAACAGCTCCTCGCCCTTGCTCACGTACATCTCCAGTCCACCACT
insert_F	TAACGCCAGGGTTTTCCCAGTCA
insert_R	CGTGCTGCTTCATGTGGTCGG

菌株 Strain	生长速率Growth rate (cm·d^-1)
菌株 Strain	PDA	5×YEG	CM
PH-1	1.20±0.05	0.83±0.10	0.85±0.10
ΔFgOAR1	1.19±0.01	0.87±0.02	0.84±0.11
ΔFgOAR1-C	1.23±0.09	0.85±0.12	0.85±0.10

菌株 Strain	生长速度Growth rate (cm·d^-1)
菌株 Strain	CM	Congo-Red	NaCl	SDS	H₂O₂
PH-1	1.20±0.05	1.15±0.10	0.98±0.11	0.54±0.15	0.75±0.11
ΔFgOAR1	1.10±0.01	0.81±0.02*	0.62±0.17*	0.37±0.09*	0.60±0.09*
ΔFgOAR1-C	1.23±0.09	1.16±0.12	0.88±0.11	0.57±0.19	0.73±0.10