基于非靶向代谢组学分析StLAC2和StLAC6差异影响玉米大斑病菌的机制

doi:10.3864/j.issn.0578-1752.2023.16.006

中国农业科学 ›› 2023, Vol. 56 ›› Issue (16): 3110-3223.doi: 10.3864/j.issn.0578-1752.2023.16.006

基于非靶向代谢组学分析StLAC2和StLAC6差异影响玉米大斑病菌的机制

邹金鹏¹^,²(), 岳浩峰², 李海笑¹^,², 刘峥³, 刘宁¹^,²(), 曹志艳¹^,²(), 董金皋¹^,²

¹ 河北农业大学植物保护学院，河北保定 071001
² 河北省植物生理与分子病理学重点实验室/华北作物改良与调控国家重点实验室，河北保定 071001
³ 保定市教育科学研究所，河北保定 071066

收稿日期:2023-05-11 接受日期:2023-06-06 出版日期:2023-08-16 发布日期:2023-08-18
通信作者:
刘宁，E-mail：lning121@126.com

曹志艳，E-mail：caoyan208@126.com
联系方式: 邹金鹏，E-mail：17338233032@163.com。
基金资助:
国家自然科学基金(31901827); 国家自然科学基金(32072370); 河北省自然科学基金(C2020204039); 河北省自然科学基金(C2021204136); 国家现代农业产业技术体系(CARS-02-25); 中央引导地方科技发展资金(226Z6502G)

Mechanism of StLAC2 and StLAC6 Differentially Affecting Setosphaeria turcica Based on Non-Targeted Metabonomics Analysis

ZOU JinPeng¹^,²(), YUE HaoFeng², LI HaiXiao¹^,², LIU Zheng³, LIU Ning¹^,²(), CAO ZhiYan¹^,²(), DONG JinGao¹^,²

¹ College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei
² Key Laboratory of Hebei Province for Plant Physiology and Molecular Pathology/State Key Laboratory of North China Crop Improvement and Regulation, Baoding 071001, Hebei
³ Academy of Educational Sciences of Baoding, Baoding 071066, Hebei

Received:2023-05-11 Accepted:2023-06-06 Published:2023-08-16 Online:2023-08-18

摘要/Abstract

摘要：

【背景】漆酶作为多酚氧化酶，在真菌生长发育及次级代谢等多方面发挥重要的作用。玉米大斑病菌（Setosphaeria turcica）基因组中含有多个漆酶基因，其中StLAC2和StLAC6对玉米大斑病菌生长发育及致病性具有差异的影响。【目的】明确StLAC2和StLAC6对玉米大斑病菌的差异作用机制，挖掘差异代谢物，为开发新的杀菌剂及病害防控新策略提供靶点。【方法】利用无缝克隆的方法将StLAC6和pHZ100-GFP质粒连接，构建StLAC6的回补表达载体。利用PEG介导的原生质体转化法，将构建好的载体转入到StLAC6基因缺失突变体的原生质体中，并利用PCR、RT-qPCR和GFP荧光验证，对所获得的阳性转化子进行鉴定，成功构建StLAC6回补菌株，并分析敲除及回补StLAC2和StLAC6对玉米大斑病菌胞内外黑色素合成及抗氧化性的影响。以野生型、StLAC2和StLAC6基因敲除突变体为试验材料，利用非靶向代谢组学分析其差异代谢物，并利用KEGG分析StLAC2和StLAC6差异作用的机制。【结果】StLAC2和StLAC6对病菌菌丝内和分泌到培养基中的黑色素合成具有差异影响，且StLAC2影响病菌的抗氧化性。代谢组分析发现与玉米大斑病菌野生型菌株相比，敲除StLAC2后无论菌丝中或分泌到培养基中的差异代谢物数量更多，KEGG分析发现差异代谢物主要为脂类尤其是磷脂，StLAC2的缺失造成多种黄酮多酚类代谢物下调。而1，8-二羟基萘型黑色素合成途径的中间代谢物小柱孢酮和柱孢酮含量在ΔStLAC2中显著增加，在ΔStLAC6中显著降低。【结论】StLAC2参与黑色素的聚合，StLAC6负调控玉米大斑病菌黑色素合成，StLAC2和StLAC6差异影响玉米大斑病菌中脂类代谢物和黑色素合成途径的中间代谢物，StLAC2缺失造成多种黄酮多酚类代谢物下调，导致抗氧化性降低。

关键词: 玉米大斑病菌, 漆酶, 代谢组学, 差异代谢物, 黑色素

Abstract:

【Background】As a polyphenol oxidase, laccase plays an important role in fungal growth, development and secondary metabolism. A plurality of laccase genes are encoded in the genome of Setosphaeria turcica, among which StLAC2 and StLAC6 have differential effects on the growth, development, and pathogenicity of S. turcica.【Objective】To clarify the differential mechanisms of StLAC2 and StLAC6 on S. turcica and explore new targets for developing new fungicides and disease control strategies by mining differential metabolites.【Method】StLAC6 was connected with pHZ100-GFP plasmid by seamless cloning, and the complementary expression vector of StLAC6 was constructed. Using PEG-mediated protoplast transformation method, the constructed vector was transferred into the protoplast of StLAC6 gene knockout mutant, and the positive transformants were identified by PCR, RT-qPCR and GFP fluorescence verification, and the StLAC6 revertant strain was successfully constructed. The effects of knocking out and reverting StLAC2 and StLAC6 on melanin synthesis and oxidation resistance in and out of S. turcica were analyzed. Taking wild-type (WT), StLAC2 and StLAC6 gene knockout mutants as experimental materials, the differential metabolites were analyzed by non-targeted metabonomics, and the mechanism of the differential action of StLAC2 and StLAC6 was analyzed by KEGG.【Result】StLAC2 and StLAC6 have differential effects on melanin synthesis in mycelium and secreted into culture medium, and StLAC2 also affects antioxidant activity of S. turcica. Metabolomic analysis found that compared with the WT strain of S. turcica, there were more differential metabolites in the mycelium or secreted into the culture medium after knocking out StLAC2, and KEGG analysis showed that the differential metabolites were mainly lipids, especially phospholipids. Meanwhile, the absence of the StLAC2 caused down-regulation of various flavonoids and polyphenols. The contents of intermediates of the 1, 8-dihydroxynaphthalene melanin biosynthesis pathway, scytalone and vermelone, significantly increased in ΔStLAC2 and decreased in ΔStLAC6.【Conclusion】The StLAC2 participates in melanin polymerization, the StLAC6 negatively regulates melanin biosynthesis in S. turcica, and the differential effects of StLAC2 and StLAC6 affect lipid metabolism and intermediates of the melanin biosynthesis pathway in S. turcica. The absence of StLAC2 caused down-regulation of various flavonoids and polyphenols, leading to decreased antioxidant activity.

Key words: Setosphaeria turcica, laccase, metabonomics, differential metabolite, melanin

邹金鹏, 岳浩峰, 李海笑, 刘峥, 刘宁, 曹志艳, 董金皋. 基于非靶向代谢组学分析StLAC2和StLAC6差异影响玉米大斑病菌的机制[J]. 中国农业科学, 2023, 56(16): 3110-3223.

ZOU JinPeng, YUE HaoFeng, LI HaiXiao, LIU Zheng, LIU Ning, CAO ZhiYan, DONG JinGao. Mechanism of StLAC2 and StLAC6 Differentially Affecting Setosphaeria turcica Based on Non-Targeted Metabonomics Analysis[J]. Scientia Agricultura Sinica, 2023, 56(16): 3110-3223.

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参考文献 32

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)
StLAC6-F	CCACCGCGGTGGCGGCCGCTCTAGAATGGTCTTTTCCATCTCAAG
StLAC6-R	CGCCCTTGCTCACCCTATCGAATTCCCGACGCAGTCCAGAGTCGC
GFP-F	GCGGCCGCCATGAGTAAAGGAGAAGAACT
GFP-R	GAATTCTGTATAGTTCATCCATGCCA
RT-StLAC6-F	TTACCGACGCAGTCCAGAGTCGC
RT-StLAC6-R	ATGGTCTTTTCCATCTCAAGGGTAG
β-tubulin-F	GCGTTTCCCTGGTCAGCTTA
β-tubulin-R	GGGAAGGGCACCATGTTG

分类 Classification	化合物 Compound	ΔStLAC2 vs WT		ΔStLAC6 vs WT
分类 Classification	化合物 Compound	FC	P value	FC	P value
二酰甘油 Diacylglycerol	CDP-DG (16:0/18:1(11Z))	0.9652	1.59E-05	1.6565	6.37E-05
	CDP-DG (16:0/18:2(9Z,12Z))	0.9751	3.32E-05	1.8412	8.23E-05
	CDP-DG (18:1(11Z)/18:1(11Z))	0.8606	4.48E-03	1.9461	4.49E-02
鞘磷脂 Sphingolipid	C17 sphingosine-1-phosphocholine	1.5874	1.64E-05	1.0236	8.45E-02
鞘磷脂 Sphingolipid	Sphingosine 1-phosphate	1.5328	3.53E-06	0.9084	9.29E-06
磷脂酰胆碱 Phosphatidyl cholines (PC and LysoPC)	PC (17:1/0:0)	2.6534	0.000814	1.0452	4.66E-03
	PC (18:0/0:0)	1.9638	3.42E-03	1.0620	0.000339
	PC (16:1(9Z)/20:4(8Z,11Z,14Z,17Z))	1.1043	5.33E-06	0.2862	8.27E-05
	PC (14:0/22:5(7Z,10Z,13Z,16Z,19Z))	0.9632	1.52E-05	0.3511	2.23E-05
磷脂酰甘油 Phosphatidyl glycerol (PG)	PG (16:0/0:0)[U]	2.4214	4.96E-05	1.1026	1.45E-03
	PG (18:0/0:0)[U]	1.5639	3.61E-02	1.0628	0.000153
	PG (18:1(9Z)/0:0)	1.8132	0.000566	1.1901	1.45E-03

基于非靶向代谢组学分析StLAC2和StLAC6差异影响玉米大斑病菌的机制

Mechanism of StLAC2 and StLAC6 Differentially Affecting Setosphaeria turcica Based on Non-Targeted Metabonomics Analysis

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