生防真菌土曲霉AT9遗传转化体系的构建及GFP标记菌株的获得

doi:10.16409/j.cnki.2095-039x.2026.02.017

摘要/Abstract

摘要： 土曲霉Aspergillus terreus可有效防治植物线虫和病原真菌，是具有良好生防潜力的丝状真菌。本研究以土曲霉AT9为材料，测试其对尖孢镰刀菌Fusarium oxysporum的抑菌作用。为探究其生防机理，首先评估渗透压稳定剂种类、酶解系统、菌龄，酶解温度、时间和转速对土曲霉原生质体制备的影响，以建立高效的土曲霉遗传转化体系。结果表明，AT9菌株培养物和发酵粗提物对尖孢镰刀菌的抑菌效果显著。AT9菌株在YES培养基中28℃培养17 h，使用0.7 mol/L KCl+0.27 mol/L CaCl₂作为渗透压稳定剂，经20 mg/mL溶壁酶+20 mg/mL蜗牛酶+20 mg/mL纤维素酶在26℃、160 r/min酶解9 h，原生质体数量最多，为2.49×10⁷cfu/mL。通过PEG介导转化法，将质粒pch-sGFP转入AT9菌株以验证该体系。结合荧光观察和PCR鉴定表明，外源基因已成功整合至AT9菌株，成功建立AT9菌株遗传转化体系，并获得绿色荧光蛋白（green fluorescent protein,GFP）标记菌株GFP-AT9。本研究成功建立了土曲霉遗传转化体系并获得GFP-AT9菌株，为解析该菌株生防机制和遗传改良提供关键的技术支撑。

关键词: 土曲霉, 生物防治, 原生质体制备, 遗传转化, GFP

Abstract: Aspergillus terreus is effective in controlling plant-parasitic nematodes and fungi, making it a filamentous fungus with good biocontrol potentiality. In this study, A. terreus strain AT9 was used as the experimental material to investigate in vitro inhibitory activity against Fusarium oxysporum. The effects of types of osmotic pressure stabilizer, enzymatic hydrolysis system, mycelial age, enzymatic digestion temperature, time and rotational speed on preparation of strain AT9 protoplasts were systematically examined. Based on these results, an efficient genetic transformation system for strain AT9 would be established and verified. The results indicated that strain AT9 culture and its crude extract from fermentation exhibited significant inhibitory effects against F. oxysporum. Strain AT9 cultured in YES medium at 28 ℃ for 17 hours yielded a maximum of 2.49×10⁷ cfu/mL protoplasts, using 0.7 mol/L KCl + 0.27 mol/L CaCl₂ as the osmotic pressure stabilizer, with enzymatic hydrolysis by20 mg/mL Lysing Enzymes, 20 mg/mL snail enzyme, and 20 mg/mL cellulolytic enzyme at 26 ℃ and 160 r/min for 9 h. Using PEG-mediated transformation, the plasmid pch-sGFP was introduced into the AT9 strain. Fluorescence observation and PCR identification confirmed that the exogenous gene had been successfully integrated into the genome of the AT9 strain, and an efficient transformation system for A. terreus AT9 was successfully established, resulting in the acquisition of a green fluorescent protein（GFP）-labeled strain GFP-AT9. This study successfully established a genetic transformation system for A. terreus and obtained the GFP-AT9 strain, providing crucial technical support for analyzing the biocontrol function and conducting genetic improvement.

Key words: Aspergillus terreus, biological control, protoplast preparation, genetic transformation, GFP

中图分类号:

S476

汪亚溪, 坚晋卓, 王丹, 刘永刚, 罗宁, 李惠霞. 生防真菌土曲霉AT9遗传转化体系的构建及GFP标记菌株的获得[J]. 中国生物防治学报, 2026, 42(3): 766-776.

WANG Yaxi, JIAN Jinzhuo, WANG Dan, LIU Yonggang, LUO Ning, LI Huixia. Construction of a Genetic Transformation System for Biocontrol Fungus Aspergillus terreus AT9 and Acquisition of GFP-Tagged Strains[J]. Chinese Journal of Biological Control, 2026, 42(3): 766-776.

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