中国农业科学

引物名称Primer name	引物序列Sequence of primers (5′-3′)	用途Usage
β-tubulin-F β-tubulin-R	GTGCGCAAGGAGGCTGAGGG CATGAAGAAATGGAGACGGGGGAA	检测β-tubulin表达量 Detect the expression level of β-tubulin
StPFA5-F StPFA5-R	GCATCATAGCCGAGTCGACA TCCTTCGACCGAGGTGTAGT	检测StPFA5表达量 Detect the expression level of StPFA5
StPFA5-Up-F StPFA5-Up-R	CTGCGCATTTGCTCTACT RATGCTCCTTCAATATCATCTTCTGGGGGTCATTTGGACGGATGT	扩增StPFA5上游片段 Amplify the upstream fragment of StPFA5
StPFA5-HPH-F StPFA5-HPH-R	ACATCCGTCCAAATGACCCCCAGAAGATGATATTGAAGGAGCAT ACGCCGAACACAGAGCAATAAAAGAAGGATTACCTCTAAACAAG	扩增HPH片段 Amplify the fragment of HPH
StPFA5-Down-F StPFA5-Down-R	CTTGTTTAGAGGTAATCCTTCTTTTATTGCTCTGTGTTCGGCGT AACCGACAACCAACCGACC	扩增StPFA5下游片段 Amplify the downstream fragment of StPFA5
G418-F G418-R	TAAAATCGACAAGCCCGGCG CAGCCCGATTTCCATTCCTC	回复转化子抗性验证 Resistance verification of complementation transformants
StMR1-F StMR1-R	ACAGCCCCCAGTTCATGAT TCTACTCGGGGTGTGTGTGTGC	检测StMR1表达量 Detect the expression level of StMR1
StPKS18-F StPKS18-R	GCGTGAGATCAATGCTGCTG TCGTTGTCGTACGTCTTGCA	检测StPKS18表达量 Detect the expression level of StPKS18
StMVP1-F StMVP1-R	GGCCTTACCGAATCACCCA CGGGCTTGTGTGTGGGATTATTG	检测StMVP1表达量 Detect the expression level of StMVP1
StSCD3-F StSCD3-R	TGTCACAGAGAATGGCTCGGTC TCCATCGTCTCTCCTCTCCAA	检测StSCD3表达量 Detect the expression level of StSCD3
St3HNR-F St3HNR-R	AAGCTCATGGACGACGTTGT ATGGCCATGCACCTGGTAAA	检测St3HNR表达量 Detect the expression level of St3HNR

引物名称Primer name	引物序列Sequence of primers (5′-3′)	用途Usage
β-tubulin-F β-tubulin-R	GTGCGCAAGGAGGCTGAGGG CATGAAGAAATGGAGACGGGGGAA	检测β-tubulin表达量 Detect the expression level of β-tubulin
StPFA5-F StPFA5-R	GCATCATAGCCGAGTCGACA TCCTTCGACCGAGGTGTAGT	检测StPFA5表达量 Detect the expression level of StPFA5
StPFA5-Up-F StPFA5-Up-R	CTGCGCATTTGCTCTACT RATGCTCCTTCAATATCATCTTCTGGGGGTCATTTGGACGGATGT	扩增StPFA5上游片段 Amplify the upstream fragment of StPFA5
StPFA5-HPH-F StPFA5-HPH-R	ACATCCGTCCAAATGACCCCCAGAAGATGATATTGAAGGAGCAT ACGCCGAACACAGAGCAATAAAAGAAGGATTACCTCTAAACAAG	扩增HPH片段 Amplify the fragment of HPH
StPFA5-Down-F StPFA5-Down-R	CTTGTTTAGAGGTAATCCTTCTTTTATTGCTCTGTGTTCGGCGT AACCGACAACCAACCGACC	扩增StPFA5下游片段 Amplify the downstream fragment of StPFA5
G418-F G418-R	TAAAATCGACAAGCCCGGCG CAGCCCGATTTCCATTCCTC	回复转化子抗性验证 Resistance verification of complementation transformants
StMR1-F StMR1-R	ACAGCCCCCAGTTCATGAT TCTACTCGGGGTGTGTGTGTGC	检测StMR1表达量 Detect the expression level of StMR1
StPKS18-F StPKS18-R	GCGTGAGATCAATGCTGCTG TCGTTGTCGTACGTCTTGCA	检测StPKS18表达量 Detect the expression level of StPKS18
StMVP1-F StMVP1-R	GGCCTTACCGAATCACCCA CGGGCTTGTGTGTGGGATTATTG	检测StMVP1表达量 Detect the expression level of StMVP1
StSCD3-F StSCD3-R	TGTCACAGAGAATGGCTCGGTC TCCATCGTCTCTCCTCTCCAA	检测StSCD3表达量 Detect the expression level of StSCD3
St3HNR-F St3HNR-R	AAGCTCATGGACGACGTTGT ATGGCCATGCACCTGGTAAA	检测St3HNR表达量 Detect the expression level of St3HNR

棕榈酰转移酶StPFA5参与调控玉米大斑病菌生长发育及致病力

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袁伟 , 张图 , 董广恩 , 石金卓 , 李海笑 , 曹志艳 , 刘宁 , 董金皋

中国农业科学 | 植物保护 2026,59(16): 3577-3590

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中国农业科学 |植物保护 2026 , 59 (16) : 3577 -3590

棕榈酰转移酶StPFA5参与调控玉米大斑病菌生长发育及致病力

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袁伟, 张图, 董广恩, 石金卓, 李海笑, 曹志艳, 刘宁, 董金皋

作者信息

河北农业大学植物保护学院/河北省植物生理与分子病理学重点实验室，河北保定 071000

通讯作者:

刘宁，E-mail：lning121@126.com。

曹志艳，E-mail：caoyan208@126.com。

作者简介:

袁伟，E-mail：19503278706@163.com。

Palmitoyl Transferase StPFA5 Regulates the Growth, Development and Pathogenicity of Setosphaeria turcica

Wei YUAN, Tu ZHANG, GuangEn DONG, JinZhuo SHI, HaiXiao LI, ZhiYan CAO, Ning LIU, JinGao DONG

Affiliations

College of Plant Protection, Hebei Agricultural University/Hebei Key Laboratory of Plant Physiology and Molecular Pathology, Baoding 071000, Hebei

出版时间: 2026-08-16 doi: 10.3864/j.issn.0578-1752.2026.16.008

文章导航

摘要

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【目的】棕榈酰化修饰是一种动态可逆的蛋白质翻译后修饰，在信号转导、代谢稳态、蛋白定位与运输等生物过程中发挥着关键的调控作用，但在玉米大斑病菌（Setosphaeria turcica）中的功能及其对病菌致病力的影响尚不清楚。研究棕榈酰转移酶StPFA5在玉米大斑病菌生长发育及致病力调控中的作用，为阐明棕榈酰化修饰介导的玉米大斑病菌生长发育与致病过程的分子机制打下基础。【方法】利用同源比对鉴定StPFA5并构建进化树，通过RT-qPCR测定StPFA5在病菌侵染过程中的表达水平。通过同源重组技术创制StPFA5的敲除突变体及回补菌株，针对菌株WT、∆StPFA5、C.∆StPFA5，测定生长速率及致病力，观察菌丝形态和菌丝萌发过程，并通过酸沉碱溶提取菌丝黑色素，RT-qPCR测定黑色素合成相关基因表达水平。此外，将各菌株接种在含刚果红和CFW的PDA平板上，测定StPFA5对病菌细胞壁稳定性的影响。通过蛋白组学和棕榈酰化修饰蛋白组学研究，结合数据库检索，鉴定WT和∆StPFA5中的差异棕榈酰化修饰位点及所属蛋白，进行功能分析。【结果】 StPFA5与异旋孢腔菌（Cochliobolus heterostrophus）PFA5同源性最高，达80.35%。与野生型相比，∆StPFA5的菌落生长速率降低、菌丝形态畸形、致病力显著下降，基因回补后恢复到野生型水平。同时，∆StPFA5的菌丝黑色素含量降低，黑色素合成相关基因表达显著下调，细胞壁胁迫敏感性降低。棕榈酰化修饰组学分析发现，敲除StPFA5导致448个棕榈酰化位点的修饰水平下调，下调位点所属蛋白富集在氨基酸合成代谢、碳代谢、糖酵解、细胞周期、染色体浓缩等通路。综合∆StPFA5表型结果发现，StPFA5可能通过调控这些途径相关蛋白的棕榈酰化水平影响玉米大斑病菌生长发育及致病力。【结论】棕榈酰转移酶StPFA5通过调控蛋白质的棕榈酰化修饰水平，促进玉米大斑病菌的生长发育和黑色素合成，并对该病菌的致病力发挥正向调控作用。

关键词

玉米大斑病菌 / 棕榈酰化修饰 / 棕榈酰转移酶 / StPFA5 / 黑色素 / 致病力

Abstract

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【Objective】 S-palmitoylation is a dynamically reversible post-translational protein modification that exerts a key regulatory role in biological processes such as signal transduction, metabolic homeostasis, and protein localization and transport in organisms. However, its function in Setosphaeria turcica and its impact on the pathogenicity of this fungus remain unclear. This study aimed to investigate the role of the palmitoyl transferase StPFA5 in regulating the growth, development and pathogenicity of S. turcica, and to lay a foundation for elucidating the molecular mechanisms underlying S. turcica growth and pathogenic processes mediated by S-palmitoylation.【Method】 StPFA5 was identified via homologous alignment and a phylogenetic tree was constructed. RT-qPCR was performed to determine the expression level of StPFA5 during the fungal infection process. The knockout mutant and complementation strain of StPFA5 were generated using homologous recombination technology. For the WT, ∆StPFA5 and C.∆StPFA5 strains, the growth rate and pathogenicity were determined, and hyphal morphology and hyphal germination were observed. Mycelial melanin was extracted by acid precipitation and alkaline dissolution, and the expression levels of melanin synthesis-related genes were detected by RT-qPCR. In addition, all strains were inoculated on PDA plates containing Congo red and CFW to assess the effect of StPFA5 on the cell wall integrity of S. turcica. Furthermore, proteomic and S-palmitoylomic analyses combined with database retrieval were conducted to identify differentially modified S-palmitoylation sites and their corresponding proteins in the WT and ∆StPFA5 strains, followed by functional annotation of these proteins.【Result】 StPFA5 shared the highest homology (80.35%) with PFA5 from Cochliobolus heterostrophus. Compared with the WT strain, ∆StPFA5 exhibited reduced colony growth rate, abnormal hyphal morphology and significantly decreased pathogenicity, and these phenotypic defects were restored to the WT level in the C.∆StPFA5 strain. Meanwhile, the melanin content in the mycelia of ∆StPFA5 was decreased, the expression of melanin synthesis-related genes was significantly down-regulated, and the sensitivity of ∆StPFA5 to cell wall stress was reduced. S-palmitoylomic analysis revealed that knockout of StPFA5 led to the downregulation of modification levels at 448 S-palmitoylation sites, and the proteins containing these downregulated sites were significantly enriched in biological pathways including amino acid anabolism, carbon metabolism, glycolysis, cell cycle and chromosome condensation. Combined with the phenotypic characteristics of ∆StPFA5, StPFA5 may affect the growth, development and pathogenicity of S. turcica by regulating the S-palmitoylation levels of proteins associated with these pathways.【Conclusion】 The palmitoyl transferase StPFA5 promotes the growth, development and melanin synthesis of S. turcica by regulating the S-palmitoylation modification levels of target proteins, and exerts a positive regulatory effect on the pathogenicity of this fungus.

Key words

Setosphaeria turcica / S-palmitoylation / palmitoyl transferase / StPFA5 / melanin / pathogenicity

引用本文

袁伟, 张图, 董广恩, 石金卓, 李海笑, 曹志艳, 刘宁, 董金皋. 棕榈酰转移酶StPFA5参与调控玉米大斑病菌生长发育及致病力. 中国农业科学, 2026 , 59 (16) : 3577 -3590 . DOI: 10.3864/j.issn.0578-1752.2026.16.008

Wei YUAN, Tu ZHANG, GuangEn DONG, JinZhuo SHI, HaiXiao LI, ZhiYan CAO, Ning LIU, JinGao DONG. Palmitoyl Transferase StPFA5 Regulates the Growth, Development and Pathogenicity of Setosphaeria turcica[J]. Scientia Agricultura Sinica, 2026 , 59 (16) : 3577 -3590 . DOI: 10.3864/j.issn.0578-1752.2026.16.008

正文

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0 引言

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【研究意义】玉米大斑病菌（Setosphaeria turcica）侵染导致的玉米大斑病是一种叶部病害，严重影响玉米产量和品质^[1]。棕榈酰化修饰是一种可逆的脂质翻译后修饰，动态可逆地调节蛋白质功能，在植物病原真菌致病过程中发挥重要作用^[2]。研究玉米大斑病菌中棕榈酰化修饰相关基因的功能及其对致病的调控机制，对植物病原真菌致病机理的理解具有重要意义，并可为玉米大斑病的防治提供新思路。【前人研究进展】玉米大斑病菌主要通过孢子萌发形成附着胞实现侵染^[3]。孢子接触玉米叶片后，在适宜条件下萌发形成极性生长的芽管，芽管感知寄主信号后顶端膨大，形成具有侵染能力的附着胞^[4-5]。附着胞中黑色素的积累是病菌成功侵染的关键。黑色素沉积于胞壁可使附着胞形成高膨压并产生侵染钉，侵染钉通过机械力穿透叶片表皮，促进病菌定殖并引发病害^[6-7]。蛋白质翻译后修饰是细胞调控生命活动的重要分子基础，包括磷酸化、泛素化、糖基化及脂质化等形式^[8-9]。棕榈酰化修饰是一类以棕榈酸为修饰分子的蛋白质翻译后修饰方式，依据连接方式可分为N-酰化、O-酰化和S-棕榈酰化3类^[10-11]。目前研究报道中所指的棕榈酰化修饰多为S-棕榈酰化^[12]。棕榈酰化修饰是动物中普遍存在的脂质修饰方式，目前已有上千种哺乳动物蛋白被证实存在棕榈酰化修饰，其参与调控先天免疫、炎症反应、葡萄糖和脂肪代谢等生理过程，并与多种疾病的发生发展密切相关^[13-14]。在植物中，棕榈酰化修饰同样发挥重要调控作用。研究发现，拟南芥不同组织中1 094种蛋白的1 849个半胱氨酸残基存在棕榈酰化修饰，这些蛋白参与物质运输、信号转导、代谢调控等细胞生理过程^[15-16]。此外，棕榈酰化修饰在真菌生长繁殖、环境适应及致病过程中的生物学功能也逐步被揭示。酿酒酵母（Saccharomyces cerevisiae）中Tlg1的棕榈酰化保证膜转运功能正常^[17-18]。尖孢镰孢西瓜专化型（Fusarium oxysporum f. sp. niveum）中，棕榈酰化修饰是病菌发挥毒力的必要条件^[19]。棕榈酰化修饰可调控蛋白质的亚细胞定位、分子稳定性、生物学活性及蛋白间的相互作用^[20-21]。该修饰由棕榈酰转移酶（palmitoyl acyltransferase，PAT）和酰基蛋白硫酯酶（acyl-protein thioesterase，APT）/棕榈酰蛋白硫酯酶（palmitoyl-protein thioesterase，PPT）协同调控^[22-23]。其中，棕榈酰转移酶是一类催化蛋白质棕榈酰化修饰的关键酶，其核心特征为保守的DHHC结构域^[24-25]。酵母棕榈酰转移酶Swf1修饰单跨膜蛋白SNARE近膜区半胱氨酸，Swf1缺失导致内体-高尔基体循环与囊泡运输紊乱，肌动蛋白组装、极性生长异常，此外，Swf1参与孢子壁组装，缺失影响产孢效率^[26-27]。【本研究切入点】棕榈酰转移酶是真菌致病机制中棕榈酰化调控的关键因子，明确其调控网络对解析病菌致病机制具有重要指导意义。在尖孢镰孢西瓜专化型中，不同棕榈酰转移酶基因缺失后，菌株在菌丝生长、分生孢子产生、细胞壁完整性维持、渗透胁迫耐受及致病力方面出现不同程度缺陷^[19]；UvPfa4是稻曲病菌（Ustilaginoidea virens）中关键的S-棕榈酰转移酶，通过棕榈酰化修饰MAPK通路核心激酶UvSlt2，调控稻曲病菌致病力、信号转导与自噬，是真菌致病机制中棕榈酰化调控的重要靶点^[28]。然而，相较于动植物，棕榈酰化修饰在植物病原菌中的功能及调控网络尚未明确。【拟解决的关键问题】通过研究StPFA5对玉米大斑病菌生长发育、黑色素合成和致病力的影响，并利用棕榈酰化修饰蛋白组学分析其主要修饰蛋白的分子功能和代谢途径，明确棕榈酰化在生长发育及侵染过程中的作用，为探讨病害防治的新方法提供支持。

1 材料与方法

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试验于2024年3月至2026年2月在河北农业大学植物保护学院/河北省植物生理与分子病理学重点实验室完成。

1.1 菌株及培养条件

以玉米大斑病菌01-23为野生型（WT）菌株，获得缺失突变体∆StPFA5和C.∆StPFA5菌株，在25 ℃的PDA培养基上进行黑暗培养。

1.2 玉米大斑病菌PFA5蛋白的鉴定和序列分析

从NCBI（https://www.ncbi.nlm.nih.gov/）检索并下载稻瘟病菌（Magnaporthe oryzae）、禾谷镰孢（Fusarium graminearum）、稻曲病菌、番茄匍柄霉（Stemphylium lycopersici）、异旋孢腔菌（Cochliobolus heterostrophus）、烟曲霉（Aspergillus fumigatus）、交链格孢（Alternaria alternata）和酿酒酵母的PFA5氨基酸序列，并进行多序列比对；比对使用Clustal Omega网站（https://www.ebi.ac.uk/jdispatcher/msa/ clustalo）与jalview软件；采用MEGA 11软件构建系统发育树^[29]；通过NCBI网站获得StPFA5蛋白序列，并通过SMART网站（https://smart.embl-heidelberg.de/）对其编码的氨基酸序列及结构域进行分析。

1.3 基因相对表达水平分析

将浓度为1×10⁴/mL WT的孢子悬浮液接种到玉米自交系B73的叶片上。分别收集接种病菌2、4、6、8 d的B73叶片的病健交界处进行RNA提取，之后用TransScript^® One-Step gDNA Removal and cDNA Kit（TransGen Biotech，北京）进行反转录。反转录完成后，采用cDNA 2 μL、引物各0.2 μL、荧光定量Mix 5 μL、H₂O 2.8 μL的体系进行RT-qPCR反应，所用内参为β-tubulin（表1），公式为2^-∆∆Ct，检测StPFA5在侵染期的表达水平。每组试验重复3次。

将菌株WT、∆StPFA5接种于PD液体培养基，25 ℃黑暗培养7 d。收集菌丝提取RNA，之后用TransScript^® One-Step gDNA Removal and cDNA Kit（TransGen Biotech，北京）进行反转录。反转录完成后，采用cDNA 2 μL、引物各0.2 μL、荧光定量Mix 5 μL、H₂O 2.8 μL的体系进行RT-qPCR反应，所用内参为β-tubulin，公式为2^-∆∆Ct，检测WT、∆StPFA5中黑色素合成相关基因StMR1、StPKS18、St3HNR、StSCD3、StMVP1（表1）的表达水平。每组试验重复3次。

1.4 StPFA5敲除突变体及回补菌株创制

分别扩增StPFA5的上下游同源臂和带有部分同源臂的潮霉素片段。将StPFA5的上游和下游与潮霉素片段连接，构建StPFA5基因敲除融合片段，通过PEG介导法转化WT菌株的原生质体，获得基因敲除突变体。用潮霉素抗性筛选基因敲除突变体，并通过PCR进行验证。

为了进行互补菌株创制，以WT株的DNA为模板，扩增StPFA5的全长。用T4连接酶将StPFA5片段连接至pHZ100载体，构建pHZ100-StPFA5载体。然后，PEG介导法将其转化到∆StPFA5菌株的原生质体中，并进行PCR验证。

1.5 表型及生长速率测定

将WT、∆StPFA5、C.∆StPFA5菌盘分别置于PDA培养基上25 ℃黑暗培养7 d，测量菌落生长直径，观察菌落形态，每菌株进行3次重复。

1.6 菌丝形态观察及附着胞形成测定

将WT、∆StPFA5、C.∆StPFA5菌株在PDA上培养7 d，制备菌丝体悬浮液。取20 μL菌丝悬浮液接种于水琼脂平板培养膜，在25 ℃黑暗培养。取100个菌丝体，在0、12、24、48 h用光学显微镜观察菌丝体附着胞的形成，每组试验重复3次。

1.7 黑色素含量检测

将WT、∆StPFA5、C.∆StPFA5的菌丝体培养7 d后，通过酸沉碱溶的方法进行黑色素提取^[30]。用450 nm处的吸光度进行定量分析，研究StPFA5对黑素合成的影响，每组试验重复3次。

1.8 致病力分析

将WT、∆StPFA5、C.∆StPFA5分别接种于PDA培养基上进行致病力测定，培养7 d后刮下菌丝体，用组织研磨仪研磨，制成浓度一致的菌丝体悬浮液，取10 μL菌丝悬浮液接种于玉米B73叶片上，25 ℃培养7 d，每组试验重复3次。

1.9 细胞壁敏感性测定

将WT、∆StPFA5、C.∆StPFA5接种在含有100 μg·mL^-1刚果红（CR）和15 μg·mL^-1植物细胞壁钙荧光白（CFW）的PDA培养基中，25 ℃黑暗培养7 d，用交叉法测定菌落直径，计算抑制率，每组试验重复3次。

1.10 蛋白组学分析

收集WT、∆StPFA5菌丝液氮研磨，加入SDT蛋白裂解液，沸水浴3 min，超声破碎后离心取上清，BCA法进行蛋白定量。每组样品各取20 μg蛋白进行SDS-PAGE凝胶电泳。每个样品取等量蛋白进行FASP酶解（filter aided sample preparation）。酶解后的肽段使用C18固相萃取小柱脱盐，脱盐后的肽段干燥后用0.1%甲酸复溶。每个样品取适量肽段，使用Vanquish Neo UHPLC system（Thermo Scientific）进行色谱分离。样品进样到Trap Column（PepMap Neo 5 µm C18 300 µm×5 mm，Thermo Scientific）后经过色谱分析柱（μPAC Neo High Throughput column，Thermo Scientific）进行梯度分离。肽段分离后用Orbitrap Astral Zoom质谱仪（Thermo Scientific）进行DIA（数据非依赖采集）质谱分析。

1.11 棕榈酰化修饰蛋白组学分析

收集WT、∆StPFA5菌丝进行液氮研磨，加入裂解缓冲液，冰浴超声破碎后离心取上清，BCA法进行蛋白定量。每种样品各取20 μg蛋白进行SDS-PAGE凝胶电泳。每种样品取等量蛋白加入N-乙基马来酰亚胺（NEM）（总浓度0.1 mol·L^-1）4 ℃孵育过夜。三氯乙酸-丙酮沉淀法去除多余的NEM，并将蛋白质沉淀物重新溶解在PBS中。然后将溶于DMSO的生物素-HPDP加入样品中，25 ℃下孵育1.5 h。用5K超滤管将每份样品的蛋白质浓缩至150 μL，用6倍冷丙酮沉淀过夜。加入适量的胰蛋白酶，在37 ℃下进行酶解过夜。之后将多肽溶解在200 µL上样/洗涤缓冲液（0.2%十二烷基硫酸钠、0.2%Triton X-100和500 mmol·L^-1 NaCl）中，并加入100 µL链霉亲和素磁珠，黑暗中翻转反应2 h，洗去非特异性吸附的蛋白质。用0.5 mmol·L^-1 Tris（2-carboxyethyl）phosphine（TCEP）对S-棕榈酰化肽段进行洗脱，然后用C18固相萃取小柱进行脱盐处理，最后用0.1%甲酸溶解。使用Vanquish Neo UHPLC色谱系统联合Orbitrap Astral Zoom质谱进行肽段分离及检测分析。将肽段进样到Trap Column（PepMap Neo 5 µm C18 300 µm×5 mm，Thermo Scientific）后经过色谱分析柱（μPAC Neo High Throughput column，Thermo Scientific）进行梯度分离。采用EASY-Spray源进行DIA（数据非依赖采集）质谱分析。

1.12 棕榈酰化修饰蛋白组学数据处理

为了消除蛋白质丰度对棕榈酰化修饰位点表达水平的潜在干扰，对蛋白组学和棕榈酰化修饰组学原始数据进行标准化预处理。首先，对两组数据分别执行质量控制与归一化：剔除在任意试验组中检测阳性率低于50%的蛋白或修饰位点；采用中位数归一化法校正样本间的系统性偏差：计算每个样本的中位数，确定所有样本中位数的全局中位数，将其作为基准计算各样本的归一化因子，并对原始信号进行校正。对于归一化后数据中的缺失值，采用数据集内非零最小值的一半进行填充。随后，进行去本底化处理，将归一化后的修饰位点信号强度除以其对应蛋白的信号强度，获得消除蛋白表达背景影响的归一化修饰水平，用于后续的差异分析。

1.13 生物信息学分析

LC-MS/MS数据采用Spectronaut软件进行DIA的蛋白质鉴定与定量分析。数据库搜索以UniProt_Setosphaeria turcica蛋白质数据库（https:// www.uniprot.org/proteomes/UP000016935）为检索基础，采用Target-Reverse数据库模式进行假阳性率控制。通过Motif-X软件（5.0.2版本）鉴定并绘制显著基序的序列标识图；利用DAVID数据库和STRING软件（10.5版本）开展Gene Ontology（GO）功能注释；依据Kyoto Encyclo-pedia of Genes and Genomes（KEGG）数据库对代谢通路进行层级分类；采用WoLF PSORT软件（0.2版本， https://wolfpsort.hgc.jp）预测蛋白质的亚细胞定位。

1.14 数据处理与分析

试验原始数据采用Excel 2021完成初步整理，使用GraphPad Prism 9软件开展统计分析与可视化。仅两组处理间指标比较采用独立样本t检验；3组及以上多处理组整体差异采用单因素方差分析（one-way ANOVA），组间两两多重比较采用Tukey HSD法。所有定量数据以均值±标准误表示，每组生物学重复n≥3。以P<0.05作为差异具有统计学意义的判定标准，P<0.01表示差异极显著。

2 结果

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2.1 StPFA5系统发育分析及其在侵染过程中的表达

对来自稻瘟病菌、禾谷镰孢、稻曲病菌、番茄匍柄霉、异旋孢腔菌、烟曲霉、交链格孢和酿酒酵母PFA5蛋白的氨基酸序列进行分析，并进一步构建系统发育树。结果显示，StPFA5（SETUDRAFT_163432）与异旋孢腔菌、番茄匍柄霉、链格孢中的直系同源物形成一个分支，而禾谷镰孢、稻瘟病菌、烟曲霉、稻曲病菌中的PFA5蛋白聚集在单独的分支中（图1-A），其中，StPFA5与异旋孢腔菌的PFA5同源性最高，为80.35%，而与酿酒酵母PFA5的同源性仅为24.45%。进一步解析StPFA5的功能结构域，发现其结构域为DHHC半胱氨酸富集域（图1-B）。

为了明确StPFA5在玉米大斑病菌侵染过程中是否发挥作用，通过RT-qPCR检测玉米大斑病菌侵染玉米叶片过程中StPFA5的表达量。结果表明，在病菌侵染过程中StPFA5存在表达，且表达水平相对较高。其中，感染2—4 d，StPFA5表达水平上升，4 d达到高峰，随后表达水平逐渐降低（图1-C）。

2.2 StPFA5敲除及回补菌株创制

为了研究StPFA5在玉米大斑病菌中的功能，采用同源重组的策略（图2-A），利用原生质体转化的方法进行StPFA5敲除突变体创制，并通过PCR对纯化的突变体进行筛选和验证（图2-B、2-C），创制得到2个缺失突变体（∆StPFA5-1和∆StPFA5-2）。此外，同样通过同源重组的策略（图2-D），创制了StPFA5敲除突变体的回补菌株，通过PCR（图2-E、2-F）、RT-qPCR（图2-G）筛选和验证，获得C.∆StPFA5-1和C.∆StPFA5-2。

2.3 StPFA5影响病菌生长及致病力

将WT、∆StPFA5、C.∆StPFA5接种至PDA平板，25 ℃培养7 d。与WT相比，∆StPFA5的生长速率变慢，C.∆StPFA5生长速率与WT相近。光学显微镜观察菌丝形态发现，∆StPFA5出现菌丝膨大、间隔变短，而C.∆StPFA5的菌丝形态与WT相似（图3-A）。

将WT、∆StPFA5、C.∆StPFA5分别接种于玉米B73叶片，测定StPFA5对玉米大斑病菌致病力的影响。结果显示，接种WT和C.∆StPFA5的玉米叶片产生明显梭状病斑，而接种∆StPFA5的玉米叶片形成的病斑较小（图3-B）。

2.4 StPFA5对玉米大斑病菌附着胞形成的影响

对培养12、24和48 h的WT、∆StPFA5、C.∆StPFA5菌丝进行萌发和附着胞形成观察，萌发进程分为菌丝、芽管形成、附着胞形成和侵染钉形成4个阶段。与WT和C.∆StPFA5相比，∆StPFA5的附着胞发育延迟。在12 h时，38% WT、42% C.∆StPFA5-1、40% C.∆StPFA5-2已萌发形成芽管，而∆StPFA5-1和∆StPFA5-2仅有19%、20%达到这一阶段。至24 h，78% WT、75% C.∆StPFA5-1、76% C.∆StPFA5-2已萌发形成芽管，而∆StPFA5-1、∆StPFA5-2均仅有50%达到这一阶段，此外，18% WT和13% C.∆StPFA5-1、16% C.∆StPFA5-2形成成熟的附着胞，而∆StPFA5无成熟的附着胞形成。48 h，∆StPFA5有成熟的附着胞形成，但无侵染钉产生，而WT和C.∆StPFA5各有8%、4%侵染钉形成（图4）。

2.5 敲除StPFA5影响黑色素合成

提取WT、∆StPFA5、C.∆StPFA5菌丝胞内黑色素发现，∆StPFA5黑色素含量低于WT，而C.∆StPFA5恢复到WT水平（图5-A）。鉴于StPFA5影响玉米大斑病菌黑色素合成，进一步测定了∆StPFA5菌株中黑色素合成相关基因的表达情况，结果显示，相比WT，∆StPFA5中参与黑色素合成的基因StMR1、StPKS18、St3HNR、StSCD3、StMVP1均表达下调（图5-B）。

黑色素对维持细胞壁的稳定性具有重要作用，为了测定StPFA5对玉米大斑病菌细胞壁稳定性的影响，在含100 μg·mL^-1刚果红（几丁质抑制剂）、15 μg·mL^-1 CFW（几丁质抑制剂）的PDA平板培养WT、∆StPFA5、C.∆StPFA5菌株，测定其生长速率。结果显示，与WT和C.∆StPFA5相比，∆StPFA5对刚果红、CFW的敏感性降低（图5-C）。

2.6 StPFA5修饰棕榈酰化位点鉴定

使用亲和定向的MS方法进行棕榈酰化位点鉴定，并对得到的棕榈酰化修饰组学数据进行去蛋白组本底处理，以排除蛋白本身表达量变化对修饰位点鉴定的影响。数据密度分布显示，∆StPFA5与WT两组所有样本均呈现单峰正态分布，重复性良好，数据质量可靠（图6-A）。鉴定得到∆StPFA5和WT棕榈酰化显著差异位点1 719个，其中1 271个上调，448个下调（图6-B、6-C）。

2.7 StPFA5修饰棕榈酰化蛋白功能分析

对∆StPFA5中下调的棕榈酰化位点所属蛋白进行GO功能富集，结果显示，生物学过程层面（图7-A）差异蛋白广泛参与细胞基础生命活动与物质代谢调控，高度聚焦于氨基酸生物合成、有机酸代谢等代谢过程。同时，在染色体浓缩等细胞周期过程存在富集。此外，差异蛋白参与谷胱甘肽代谢通路。细胞组分层面（图7-A、7-B），差异蛋白的空间分布高度集中在细胞质，其次是膜结构和线粒体。分子功能层面（图7-A），差异蛋白的核心活性聚焦于催化反应与核苷酸/ATP结合。

KEGG通路富集分析显示，差异显著性前30的通路条目中，差异蛋白主要富集在代谢过程，如氨基酸合成代谢、碳代谢及辅因子生物合成，以及糖酵解、丙酮酸代谢等代谢过程。此外，差异蛋白在细胞周期、减数分裂、泛素介导的蛋白水解、核质运输等通路也存在富集（图7-C）。

3 讨论

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3.1 棕榈酰化修饰影响玉米大斑病菌的生长发育及致病力

棕榈酰化修饰是细胞维持胞内平衡和对细胞外刺激作出充分应答的重要分子机制^[31-32]。近年来，棕榈酰化修饰在植物病原真菌中的调控作用被逐渐揭示^[33]。在稻曲病菌中，棕榈酰转移酶UvPfa3和UvPfa4是调控菌丝极性生长的关键因子，敲除UvPfa3或UvPfa4后，病菌菌丝生长速率显著减慢，且表现出菌丝顶端膨大、分枝增多且紊乱^[28]。本研究创制了棕榈酰转移酶编码基因StPFA5的敲除突变体（∆StPFA5）和回补菌株（C.∆StPFA5），对野生型、∆StPFA5和C.∆StPFA5菌株进行菌落生长速率测定，结果显示，与野生型相比，∆StPFA5生长速率减慢，C.∆StPFA5的生长速率恢复至与野生型相近水平，进一步观察菌丝形态发现，StPFA5的敲除导致病菌菌丝形态畸形膨大。棕榈酰化修饰组学分析结果显示，相比野生型，∆StPFA5中大量代谢相关蛋白棕榈酰化水平显著下调。综上推测，StPFA5的缺失导致细胞代谢异常，破坏了真菌菌丝顶端极性生长的物质供应，进而降低细胞分裂效率、阻碍菌丝延伸，最终导致菌落生长速率下降、菌丝形态畸形。

细胞周期的精准调控是植物病原真菌完成孢子萌发、侵染结构分化及宿主定殖的前提^[34-35]。已有研究表明，尖孢镰孢西瓜专化型中，棕榈酰转移酶FonPAT2可通过棕榈酰化修饰细胞周期调控因子AP-2复合体，保障病菌致病力的正常发挥^[19]。本研究中，对野生型、∆StPFA5和C.∆StPFA5的致病力进行测定发现，StPFA5的敲除降低了玉米大斑病菌的致病力。进一步测定菌丝萌发速率发现，相比野生型和C.∆StPFA5，∆StPFA5菌丝萌发速率变慢。此外，棕榈酰化修饰组学结果显示，相比野生型，∆StPFA5中大量细胞周期相关蛋白的棕榈酰化水平显著下调。综上推测，StPFA5通过修饰细胞周期相关蛋白的棕榈酰化水平，影响玉米大斑病菌菌丝萌发速率和侵染结构附着胞的形成，进而调控病菌的致病力。

3.2 StPFA5调控的棕榈酰化修饰通过参与玉米大斑病菌的黑色素合成影响病菌致病力

真菌黑色素是一类高分子量、非均质、暗褐色至黑色的生物色素，是真菌长期进化过程中形成的重要次生代谢产物。黑色素在真菌适应极端环境、抵御外界胁迫及侵染宿主等生理过程中发挥着不可替代的作用^[36-37]。根据合成前体及代谢途径的差异，真菌黑色素主要分为二羟萘（DHN）型和多巴（L-DOPA）型^[38]。其中，植物病原真菌以合成DHN型黑色素为主。黑色素是植物病原真菌的关键毒力因子^[39]，玉米大斑病菌、稻瘟病菌的附着胞中积累的黑色素可形成致密屏障以维持附着胞内高膨压，为侵入钉穿透叶片表皮提供机械动力。若黑色素合成受阻，病原菌侵染能力则完全丧失^[40-41]。

已有研究表明，在烟曲霉中，棕榈酰化修饰黑色素合成关键酶聚酮合酶Alb1和还原酶ArpR1/2，通过调控这些酶膜定位、活性与稳定性，影响真菌黑色素合成与致病力^[42-43]。本研究中，StPFA5的缺失导致玉米大斑病菌形成病斑面积变小，致病力下降，进一步分析发现，相比野生型，∆StPFA5的菌丝黑色素含量减少，推测玉米大斑病菌致病力下降与黑色素含量减少有关。为探究黑色素含量减少的调控机制，通过RT-qPCR测定∆StPFA5中黑色素合成相关基因的表达水平，结果显示，相比野生型，∆StPFA5中黑色素合成相关基因StMR1、StPKS18、St3HNR、StSCD3、StMVP1均表达下调。进一步分析棕榈酰化修饰组学结果发现，∆StPFA5中黑色素合成相关基因棕榈酰化水平无明显变化，但大量碳代谢、糖酵解和丙酮酸代谢过程的相关蛋白棕榈酰化水平显著下调。而DHN-黑色素的核心前体是乙酰-CoA/丙二酰-CoA，由中心碳代谢（糖酵解→丙酮酸→乙酰-CoA）持续供应^[44]。综上推测，StPFA5通过修饰碳代谢、糖酵解和丙酮酸代谢相关蛋白的棕榈酰化水平调控玉米大斑病菌黑色素的合成，进而影响病菌致病力。

4 结论

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棕榈酰转移酶StPFA5的突变导致玉米大斑病菌生长速率变慢，菌丝形态畸形，黑色素含量减少，病菌致病力降低。StPFA5通过调控蛋白棕榈酰化水平在玉米大斑病菌的生长、黑色素合成及病菌致病力中发挥重要作用。

基金

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国家重点研发计划(2023YFD1401500)
河北省自然科学基金(C2024204044)
河北省自然科学基金(C2025204211)
国家玉米产业技术体系(CARS-02-30)

参考文献

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文献

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2026年第59卷第16期

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doi: 10.3864/j.issn.0578-1752.2026.16.008

接收时间：2026-03-16
首发时间：2026-09-03
出版时间：2026-08-16

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收稿日期：2026-03-16
录用日期：2026-04-16

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国家重点研发计划(2023YFD1401500)

河北省自然科学基金(C2024204044)

河北省自然科学基金(C2025204211)

国家玉米产业技术体系(CARS-02-30)

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河北农业大学植物保护学院/河北省植物生理与分子病理学重点实验室，河北保定 071000

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曹志艳，E-mail：caoyan208@126.com。

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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