ABC转运蛋白基因LeABC2在香菇耐热性中的功能研究

doi:10.16420/j.issn.0513-353x.2025-0545

园艺学报 ›› 2026, Vol. 53 ›› Issue (5): 1315-1328.doi: 10.16420/j.issn.0513-353x.2025-0545

• 遗传育种·种质资源·分子生物学 • 上一篇下一篇

ABC转运蛋白基因LeABC2在香菇耐热性中的功能研究

吴玉龙¹, 王艺菲¹, 刘高磊¹^,², 王璐¹, 赵之栋¹, 王卓仁³, 肖扬¹^,^*()

¹ 华中农业大学应用真菌研究所，农业微生物资源发掘与利用全国重点实验室，湖北洪山实验室，武汉 430070
² 武汉市农科院蔬菜研究所，武汉 430065
³ 湖北省农业科学院经济作物研究所，武汉 430064

收稿日期:2025-11-13 修回日期:2025-12-01 出版日期:2026-05-25 发布日期:2026-05-26
通讯作者:
^* E-mail：xiaoyang@mail.hzau.edu.cn
基金资助:
湖北省支持种业高质量发展资金项目(HBZY2023B004-2); 现代农业产业技术体系建设专项资助(CARS-20)

Study on the Function of ABC Transporter Gene LeABC2 in Thermotolerance of Lentinula edodes

WU Yulong¹, WANG Yifei¹, LIU Gaolei¹^,², WANG Lu¹, ZHAO Zhidong¹, WANG Zhuoren³, XIAO Yang¹^,^*()

¹ Institute of Applied Mycology/State Key Laboratory of Agricultural Microbiology/Hubei Hongshan Laboratory， Huazhong Agricultural University， Wuhan 430070， China
² Institute of Vegetable， Wuhan Academy of Agricultural Sciences and Technology， Wuhan 430065， China
³ Institute of Economic Crops， Hubei Academy of Agricultural Sciences， Wuhan 430064， China

Received:2025-11-13 Revised:2025-12-01 Published:2026-05-25 Online:2026-05-26
Contact:
^* E-mail：xiaoyang@mail.hzau.edu.cn

摘要/Abstract

摘要：

测定了126个香菇菌株的耐热性表型，经全基因组关联分析，挖掘出与香菇耐热性相关的遗传位点，发现ABC转运蛋白基因LeABC2与香菇耐热性密切相关。通过RNAi技术沉默LeABC2基因后，转化子菌株在高温胁迫后恢复生长速度显著提升，临界耐热温度较野生型菌株提高。研究结果表明，LeABC2基因对香菇菌丝耐热性有负调控作用。

关键词: 香菇, 高温胁迫, 耐热性, ABC转运蛋白, 全基因组关联分析

Abstract:

In this study，thermotolerance phenotypes of 126 Lentinula edodes strains were evaluated. Through genome-wide association analysis，genetic loci associated with thermotolerance in L. edodes were identified. Further investigation revealed a significant correlation between the ABC transporter gene LeABC2 and thermotolerance in L. edodes. Upon silencing the LeABC2 gene via RNAi，the recovery growth rates of the transformant strains after high-temperature stress were significantly enhanced，and the critical high-temperature thresholds were higher compared to the wild-type strain. These results indicated that the LeABC2 gene negatively regulates the mycelial thermotolerance of L. edodes.

Key words: Lentinula edodes, high temperature stress, thermotolerance, ABC transporter, genome-wide association study

吴玉龙, 王艺菲, 刘高磊, 王璐, 赵之栋, 王卓仁, 肖扬. ABC转运蛋白基因LeABC2在香菇耐热性中的功能研究[J]. 园艺学报, 2026, 53(5): 1315-1328.

WU Yulong, WANG Yifei, LIU Gaolei, WANG Lu, ZHAO Zhidong, WANG Zhuoren, XIAO Yang. Study on the Function of ABC Transporter Gene LeABC2 in Thermotolerance of Lentinula edodes[J]. Acta Horticulturae Sinica, 2026, 53(5): 1315-1328.

图/表 11

图1 香菇耐热性系数的测定方法

Fig. 1 Method for determining the thermotolerance coefficient of Lentinula edodes

表1 本研究所使用的引物

Table 1 Primer sequences used in this study

引物名称 Primer name	引物序列（5′-3′） Primer sequence
ABC-DNA-F	TGGCTGTGGACAGTCTCTACAGTAT
ABC-DNA-R	GTCTAATACTACTTTAGAAC
ABC-cDNA-F	TGGCTGTGGACAGTCTCTACA
ABC-cDNA-R	GTCTAATACTACTTTAGAACG
ABC-RNAi-F	attgactgcttgaatggtaccCACACGAGGGCTCAGTCGC
ABC-RNAi-R	ctcgcagctcttcacgaattcGCAGTTTGCTGCAGACCCA
Leactin-F	ccacctcaaacttcggaattcGCAGTATTTATACCTACGGAGCGG
Leactin-R	tcttccgag CGTGAAGAGCTGCGAGTGTTG
Legpd-F	tgccactggcTGAAAAGACATGGATTGAGCCA
Legpd-R	tctagaggatccccgggTACCGTACATCCCTTGCTCTGC
qPCR-F	TGGTATTGGACCGTTGGATTT
qPCR-R	CGGCGAAAGAGACATAGTAGAAG
Leactin-qPCR-F	GCATCCTGTCCTTCTTACCGAG
Leactin-qPCR-R	AAGAGCGAAACCCTCGTAGATG
GPD-F	TTCGTTTCAGGCCTTGCCTC
HYG-R	CGGTGTCGTCCATCACAGTT

图2 重组质粒LeABC2-RNAi载体图谱

Fig. 2 Vector map of the recombinant plasmid LeABC2-RNAi

表2 香菇10个代表性菌株菌丝体在不同高温下的耐热系数（R）

Table 2 Mycelial thermotolerance coefficients among the ten representative strains of Lentinula edodes under different high temperatures

菌株 Stain	33 ℃	35 ℃	37 ℃	39 ℃	41 ℃
香430 Xiang 430	0.791	0.782	0.951	0.772	—
日丰34 Rifeng 34	0.952	0.852	0.870	0.471	—
香9 Xiang 9	0.843	0.804	0.743	0.457	—
LEQC76	0.874	0.925	0.705	0.528	—
LMLH14	0.795	0.756	0.684	0.516	—
GAN059	0.602	0.796	0.783	0.580	—
LMLHL17	0.992	0.992	0.350	0.400	—
LMLH11	0.876	0.723	0.729	0.455	—
LEQC79	0.792	0.785	0.721	0.298	—
LPZH211	0.805	0.805	0.758	0.405	—
均值Mean	0.832	0.822	0.729	0.488	—
标准差 Standard deviation	0.101	0.077	0.148	0.120	—

图3 126个香菇菌株在37 ℃高温胁迫下的耐热系数分布图

Fig. 3 The frequency distribution of the thermotolerance coefficients of the 126 Lentinula edodes strains under 37 ℃ high temperature stress

图4 126 个香菇菌株耐热性状的全基因组关联分析 A：耐热性关联SNP的曼哈顿图。黑色虚线是全基因组显著阈值（P = 9.03 × 10-7，Zhang et al.，2002），用于筛选与性状强关联的位点，箭头所指为基因组第34 Scaffolds上的SNP（位置：34：208168），显著高于全基因组阈值，提示其与耐热性高度相关。B：分位数—分位数（QQ）图。对角线（虚线）为理想情况下，若所有SNP与性状无关联，观测值应沿对角线分布

Fig. 4 Genome-wide association study of the thermotolerance traits in the 126 Lentinula edodes strains A：Manhattan plot of SNPs associated with thermotolerance. The black horizontal dashed line indicates the genome-wide significance threshold（P = 9.03 × 10-7，Zhang et al.，2002）used to screen for loci strongly associated with the trait. The arrow points to a SNP on Scaffold_34（position：34：208168）whose value significantly exceeded the genome-wide threshold，suggesting a strong association with thermotolerance. B：Quantile-quantile（Q-Q）plot. The diagonal dashed line represents the expected distribution of observed values if no SNPs were associated with the trait. The deviation of observed points from this line indicates potential trait-associated SNPs

图5 LeABC2的基因结构（A）和ABC2蛋白的系统发育树（B）

Fig. 5 Gene structure of LeABC2（A）and phylogenetic tree of the ABC2 protein（B）

图6 重组质粒LeABC2-RNAi载体图谱及验证 M1：BM2000 + DNA Marker；1 ~ 8：阳性RNAi大肠杆菌转化子

Fig. 6 Vector map and identification of the recombinant plasmid LeABC2-RNAi M1：BM2000 + DNA Marker；1-8：Positive RNAi transformants of E. coli

图7 转化子PCR鉴定 A：阳性农杆菌菌落PCR鉴定（M1：BM2000+ DNA Marker；1 ~ 12：阳性RNAi农杆菌转化子）；B：用引物GPD-F和HYG-R鉴定RNAi转化子基因片段（M1：BM 2000+ DNA Marker；1 ~ 10：RNAi转化子RNAi-1 到 RNAi-10；11：LeABC2-RNAi质粒作为阳性对照；12：S606的DNA作为阴性对照；13：空载对照DNA作为阴性对照；14：无菌水作为阴性对照）

Fig. 7 PCR identification of the transformants A：PCR identification of positive Agrobacterium colonies（M1：BM2000+ DNA marker，1-12：Positive RNAi transformants of Agrobacterium）；B：Identification of the gene fragment from RNAi transformants with primers GPD-F and HYG-R（M1：BM 2000+ DNA marker；1-10：RNAi transformants RNAi-1 to RNAi-10；11：Plasmid LeABC2-RNAi used as positive control；12：DNA from S606 used as negative control；13：DNA from control used as negative control；14：Sterile water used as negative control）

图8 qRT-PCR检测LeABC2转化子基因表达水平 S606：野生型菌株；对照：空载菌株；R-1和R-2：RNAi转化子菌株

Fig. 8 qRT-PCR determination of the relative expression level of LeABC2 in the transformants S606：Wild-type strain；Control：Control strain；R-1 and R-2：RNAi transformants ** P < 0.01

图9 不同温度热激后香菇菌株生长情况

Fig. 9 Growth conditions of Lentinula edodes strains after heat stress under different temperatures ** P < 0.01，*** P < 0.001，**** P < 0.0001

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ABC转运蛋白基因LeABC2在香菇耐热性中的功能研究

Study on the Function of ABC Transporter Gene LeABC2 in Thermotolerance of Lentinula edodes

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ABC转运蛋白基因LeABC2在香菇耐热性中的功能研究

Study on the Function of ABC Transporter Gene LeABC2 in Thermotolerance of Lentinula edodes

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