大麦条纹病菌Pg03852基因功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2025-0741

生物技术通报 ›› 2026, Vol. 42 ›› Issue (1): 262-270.doi: 10.13560/j.cnki.biotech.bull.1985.2025-0741

大麦条纹病菌Pg03852基因功能研究

马如清¹^,²(), 冯雪琪¹^,², 杨清岚¹^,², 汪军成¹^,², 孟亚雄¹^,², 马小乐¹^,², 李葆春¹^,³, 姚立蓉¹^,², 王化俊¹^,², 司二静¹^,²()

^1.干旱生境作物学国家重点实验室甘肃省作物遗传改良与种质创新重点实验室，兰州 730070
^2.甘肃农业大学农学院，兰州 730070
^3.甘肃农业大学生命科学技术学院，兰州 730070

收稿日期:2025-07-10 出版日期:2026-01-26 发布日期:2026-02-04
通讯作者: 司二静，副教授，研究方向：作物遗传育种；E-mail: siej@gsau.edu.cn
作者简介:马如清，硕士研究生，研究方向：作物遗传育种；E-mail: 2454840317@qq.com
基金资助:
国家自然科学基金项目(32160496);国家自然科学基金项目(32460517);国家自然科学基金项目(32160647);国家大麦青稞产业技术体系(CARS-05-02A-02);甘肃省教育厅产业支撑计划项目(2021CYZC-12);甘肃农业大学大学生创新创业训练项目(202501036)

Functional Analysis of Pg03852 Gene in Pyrenophora graminea

MA Ru-qing¹^,²(), FENG Xue-qi¹^,², YANG Qing-lan¹^,², WANG Jun-cheng¹^,², MENG Ya-xiong¹^,², MA Xiao-le¹^,², LI Bao-chun¹^,³, YAO Li-rong¹^,², WANG Hua-jun¹^,², SI Er-jing¹^,²()

^1.State Key Laboratory of Aridland Crop Science, Gansu Provincial Key Lab of Crop Improvement & Germplasm Enhancement, Lanzhou 730070
^2.College of Agronomy, Gansu Agricultural University, Lanzhou 730070
^3.College of Life Sciences and Technology, Gansu Agriculture University, Lanzhou 730070

Received:2025-07-10 Published:2026-01-26 Online:2026-02-04

摘要/Abstract

摘要：

目的探究Pg03852基因在大麦条纹病菌中的功能，为研究该病原菌与宿主的互作机制奠定基础。方法基于转录组数据筛选到大麦条纹病菌侵染寄主过程中高表达基因Pg03852，通过生物信息学分析、信号肽活性检测、诱导/抑制免疫反应及RNA干扰突变体制备及致病性分析等技术研究该基因的功能。结果生物信息学分析表明，Pg03852基因编码276个氨基酸，二级结构以无规则卷曲为主，占比为73.19%。Pg03852为不稳定的亲水性蛋白，无跨膜区域且无保守结构域，但具有信号肽且定位在细胞外。信号肽活性检测显示，Pg03852的信号肽转化子能够在CMD-W和YPRAA上正常生长，TTC显色进一步表明该蛋白的信号肽具有分泌功能。诱导和抑制免疫反应结果显示，Pg03852不能诱导细胞坏死，但是可抑制BAX诱导的植物组织坏死。基于PEG介导的原生质体转化法获得2个RNA干扰突变体Pg03852-RNAi-1和Pg03852-RNAi-2，RT-qPCR结果显示，相较于野生型菌株QWC，Pg03852-RNAi-1和Pg03852-RNAi-2的表达量分别下降了58.76%和48.24%，而发病率较QWC分别降低了34.67%和38.00%，且菌落生长速度均显著低于野生株QWC（P<0.05）。组织学观察发现，RNA干扰突变体的菌丝更为密集、卷曲且形态纤细。台盼蓝染色与DAB染色结果发现，干扰突变体侵染所引起的细胞死亡程度与活性氧积累水平均弱于QWC。结论 Pg03852参与大麦条纹病菌的生长发育，并在调控大麦条纹病的致病性中起关键作用。

关键词: 大麦条纹病菌, 信号肽, 免疫反应, RNA干扰, 致病力

Abstract:

Objective To explore the function of gene Pg03852 in Pyrenophora graminea, thereby establishing a crucial theoretical foundation for studying the molecular interactions between this pathogen and its host plant. Method Based on transcriptomic data, the highly expressed gene Pg03852 during the host infection process of P. graminea was identified. Preliminary functional characterization of this gene was conducted by utilizing a combination of bioinformatic analyses, signal peptide activity assays, induction/suppression of immune responses, and the generation of RNA interference (RNAi) mutants followed by pathogenicity assessment. Result Bioinformatics analysis indicated that the Pg03852 gene encoded 276 amino acids, with a secondary structure predominantly composed of disordered coils accounting for 73.19%.Pg03852 was an unstable hydrophilic protein lacking transmembrane regions and conserved domains, but possessing a signal peptide and localized to the extracellular space. Signal peptide activity assays demonstrated that the Pg03852 signal peptide transformant grew normally on CMD-W and YPRAA media. TTC staining further confirmed the secretory function of this protein’s signal peptide. Results from immune response induction and inhibition experiments showed that Pg03852 did not induce cell necrosis but suppressed BAX-induced necrosis in plant tissues. Two RNAi mutants, Pg03852-RNAi-1 and Pg03852-RNAi-2, were obtained via PEG-mediated protoplast transformation. RT-qPCR results showed that Pg03852-RNAi-1 and Pg03852-RNAi-2 had expression reductions of 58.76% and 48.24% compared to the wild-type strain QWC, respectively, with disease incidence rates of 34.67% and 38.00% lower than the wild-type strain QWC. Both mutants also demonstrated significantly slower colony growth rates than the wild-type strain QWC (P<0.05). Histological observations revealed that hyphae in the RNAi mutants were denser, more convoluted, and had a slender morphology. Trypan blue staining and DAB staining results indicated that both the extent of cell death induced by infection with the interference mutants and the levels of reactive oxygen species accumulation were weaker than those observed in wild-type strain QWC. Conclusion Pg03852 is involved in the growth and development of the P. graminea and plays a critical role in regulating its pathogenicity.

Key words: Pyrenophora graminea, signal peptide, immune response, RNA interference, pathogenicity

马如清, 冯雪琪, 杨清岚, 汪军成, 孟亚雄, 马小乐, 李葆春, 姚立蓉, 王化俊, 司二静. 大麦条纹病菌Pg03852基因功能研究[J]. 生物技术通报, 2026, 42(1): 262-270.

MA Ru-qing, FENG Xue-qi, YANG Qing-lan, WANG Jun-cheng, MENG Ya-xiong, MA Xiao-le, LI Bao-chun, YAO Li-rong, WANG Hua-jun, SI Er-jing. Functional Analysis of Pg03852 Gene in Pyrenophora graminea[J]. Biotechnology Bulletin, 2026, 42(1): 262-270.

图/表 6

表1 引物列表

Table 1 Primer list

引物 Primer	序列 Sequence（5'-3'）	用途 Purpose
Pg03852-pSuC2-F	AATTCATGAAGGTCTCACAGTTTCTCACCTGCCTCATTGCCGCGCAAGCAATCACCGCCC	构建酵母表达载体
Pg03852-pSuC2-R	TCGAGGGCGGTGATTGCTTGCGCGGCAATGAGGCAGGTGAGAAACTGTGAGACCTTCATG
Pg03852-Cal I-F	CCCATCGATATGAAGGTCTCACAGTTTCT	干扰片段克隆
Pg03852-Sma I-R	TCCCCCGGGCATGAACACACCAGCAAGAA
Pg03852-Kpn I-F	GGGGTACCTCCTCTCATCGGACAACACA
Pg03852-Bgl II-R	GAAGATCTGTTCTCGTCACAGCCACAGA
Pg03852-Xho I-F	CCGCTCGAGTCCTCTCATCGGACAACACA
Pg03852-Hind III-R	CCCAAGCTTGTTCTCGTCACAGCCACAGA
HygB-F	ATGAAAAAGCCTGAACTCAC	潮霉素鉴定
HygB-R	CTATTCCTTTGCCCTCGGA
Pg03852-qPCR-F	AGCAACACCATACAGAGCCG	实时荧光定量PCR
Pg03852-qPCR-R	GAAGCGGTAGGGGTTGTTGT	实时荧光定量PCR
PgActin-F	TGTTGACATGGCTGGTCGTGATC	内参基因
PgActin-R	TCGGCGGTGGTGGAGAAGG	内参基因

图1 Pg03852信号肽活性检测

Fig. 1 Activity assay of Pg03852 signal peptide

图2 Pg03852 诱导/抑制本氏烟叶片中坏死细胞死亡表型

Fig. 2 Pg03852 induces/inhibits the death phenotypes of necrotic cells in Baccata nicotiana leaves

图3 干扰菌株潮霉素抗性鉴定（A）及Pg03852基因的相对表达量检测（B）A： DL 15000+2 000 DNA marker；泳道1为空载体（pSilent-1）；泳道2为野生株QWC；泳道3-4为干扰突变体Pg03852-RNAi-1、Pg03852-RNAi-2。B： Pg03852的相对表达量。采用t检验，*表示在0.05水平差异显著A： Pg03852效应蛋白在本氏烟草上单独表达鉴定结果，BAX和INF1作为阳性对照，GFP作为阴性对照；B： Pg03852效应蛋白在本氏烟草上与BAX共表达鉴定结果，PVX∶BAX+PVX∶Avh240注射的烟草叶片作为阳性对照，PVX∶BAX+PVX∶GFP作为阴性对照

Fig. 3 Identification of thaumatin resistance in interfering strains (A) and detection of relative expression of the Pg03852 gene (B)A: DL 15000+2 000 DNA marker; lane 1 is the empty vector (pSilent-1); lane 2 is the wild strain QWC; lane 3-4 are the mutants Pg03852-RNAi-1 and Pg03852-RNAi-2. B. Relative expression of Pg03852. A t-test was used, and * indicates significant differences at the 0.05 levelA: Pg03852 effector protein alone was identified in this tobacco, BAX and INF1 were used as positive control, and GFP was used as negative control; B: Pg03852 effector protein co-expressed with BAX was identified in this tobacco, PVX: BAX+PVX:Avh240 injected tobacco leaves were used as positive control, and PVX: BAX+GFP was used as negative control

图4 在PDA培养基上接菌7 d的生长形态和生长速率及菌丝形态观察A： PDA培养基上接菌7 d的野生株QWC、干扰突变株Pg03852-RNAi-1、Pg03852-RNAi-2的生长形态；B：菌丝形态观察；C：菌株生长速率

Fig. 4 Growth morphology, growth rate, and hyphal morphology observed after 7 d of inoculation on PDA mediumA: Growth morphology of wild-type QWC, interference mutant strains Pg03852-RNAi-1, and Pg03852-RNAi-2 after 7 d of inoculation on PDA medium. B: Observation of mycelium morphology. C: Strain growth rate

图5 致病性分析及染色A： QWC和干扰突变体发病率统计（P<0.05）；B： QWC和干扰突变体叶绿素相对含量（P<0.05） C： QWC和干扰突变体的染色结果

Fig. 5 Pathogenicity analysis and stainingA: Statistics of QWC and interference mutant incidence (P<0.05). B: Relative chlorophyll content of QWC and interference mutants (P<0.05). C: Staining results of QWC and interference mutants

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大麦条纹病菌Pg03852基因功能研究

Functional Analysis of Pg03852 Gene in Pyrenophora graminea

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