小麦条锈菌产孢基因<i>PsCON6</i>的克隆及功能验证

doi:10.7668/hbnxb.20195885

华北农学报 ›› 2025, Vol. 40 ›› Issue (4): 185-193. doi: 10.7668/hbnxb.20195885

所属专题： 小麦；生物技术；

• 资源环境·植物保护 • 上一篇下一篇

小麦条锈菌产孢基因PsCON6的克隆及功能验证

刘贝贝¹^,², 沈煜洋², 邓菲菲², 陈江华², 李进², 李广阔², 高海峰²^,, 李月¹^,

¹ 新疆农业大学生命科学学院,新疆极端环境生物生态适应与进化重点实验室,新疆乌鲁木齐 830052
² 新疆维吾尔自治区农业科学院植物保护研究所,农业农村部西北荒漠绿洲作物有害生物综合治理重点实验室, 新疆农业生物安全重点实验室,新疆乌鲁木齐 830091

收稿日期:2025-02-28 出版日期:2025-08-28
通讯作者:
李月(1984-),女,河南许昌人,副教授,博士,主要从事作物逆境分子生物学研究。
高海峰(1983-),男,河南周口人,研究员,博士,主要从事粮食作物基因功能鉴定及分子机理解析研究。
作者简介:
刘贝贝(1998-),女,河南洛阳人,硕士,主要从事粮食作物逆境分子生物学研究。
基金资助:
新疆维吾尔自治区重点研发专项(2022B02015-3); 新疆小麦产业技术体系(XJARS-01); 现代农业产业技术体系(CARS-03-88)

Cloning and Functional Validation of the Spore Production Gene PsCON6 from Wheat Stripe Rust Fungus

LIU Beibei¹^,², SHEN Yuyang², DENG Feifei², CHEN Jianghua², LI Jin², LI Guangkuo², GAO Haifeng²^,, LI Yue¹^,

¹ College of Life Sciences,Xinjiang Agricultural University,Xinjiang Key Laboratory for Ecological Adaptation and Evolution of Extreme Environment Biology,Urumqi 830052,China
² Institute of Plant Protection,Xinjiang Academy of Agricultural Sciences,Key Laboratory of Integrated Pest Management on Crop in Northwestern Oasis,Ministry of Agriculture and Rural Affairs,Xinjiang Key Laboratory of Agricultural Biosafety,Urumqi 830091,China

Received:2025-02-28 Published:2025-08-28

摘要/Abstract

摘要：

小麦条锈病是由小麦条锈菌引起的重大病害,严重威胁我国粮食安全。夏孢子是条锈菌繁殖、传播和侵染的关键介质,但其产孢相关基因的调控机制尚不清楚。筛选并验证小麦条锈菌侵染前期高表达的候选基因PsCON6的功能,为解析其致病机制提供新线索。通过同源克隆从小麦条锈菌中获取PsCON6基因,利用qRT-PCR分析其在侵染前期的表达模式。通过生物信息学技术分析预测PsCON6蛋白的氨基酸序列、保守结构域及理化性质。通过寄主介导的基因沉默(BSMV-HIGS)技术瞬时沉默PsCON6,检测寄主活性氧面积、病原菌菌丝长度与面积及病原菌生物量。通过瞬时表达确定PsCON6蛋白的亚细胞定位。PsCON6在小麦条锈菌侵染前期显著高表达,其编码的蛋白含2个conidiation-specific protein 6保守结构域,由83个氨基酸组成。HIGS沉默PsCON6后,寄主活性氧水平显著升高,病原菌菌丝长度和面积显著减少,但孢子量未受显著影响。亚细胞定位表明,PsCON6定位于细胞膜。结果表明,PsCON6可能参与调控小麦条锈菌的菌丝生长过程,但对夏孢子形成无直接影响,其功能可能存在冗余,为揭示条锈菌致病机制提供了新靶点,并为后续深入解析其分子机制奠定了基础。

关键词: 小麦条锈菌, PsCON6, HIGS, 产孢基因

Abstract:

Wheat stripe rust,caused by Puccinia striiformis f.sp.tritici,is a major disease that severely threatens China's food security.Urediniospores are key agents for the reproduction,dissemination,and infection of the pathogen,but the regulatory mechanisms of sporulation-related genes remain unclear.This study aims to screen and validate the function of the candidate gene PsCON6,which is highly expressed during the early infection stage of P.striiformis,to provide new insights into its pathogenic mechanisms.The PsCON6 gene was obtained from P.striiformis via homologous cloning,and its expression pattern during early infection was analyzed using qRT-PCR.Bioinformatics technology analysis of the amino acid sequence,conserved domains and physicochemical properties of the PsCON6 protein.Barley Stripe mosaic virus host-induced gene silencing(BSMV-HIGS)was used to transiently silence PsCON6,followed by measurements of host reactive oxygen species accumulation,fungal hyphal length and area,and pathogen biomass.Subcellular localization of PsCON6 was determined through transient expression assays.PsCON6 was significantly upregulated during the early infection stage of P.striiformis.The encoded protein contained two conserved conidiation-specific protein 6 domains and consisted of 83 amino acids.After HIGS-mediated silencing of PsCON6,the level of reactive oxygen species in the host significantly increased,while the length and area of the fungal hyphae significantly decreased,whereas urediniospore production remained unaffected.Subcellular localization revealed that PsCON6 was localized to the cell membrane.PsCON6 likely participates in regulating hyphal growth in P.striiformis but does not directly influence urediniospore formation,suggesting potential functional redundancy.The research findings provide new targets for revealing the pathogenic mechanism of wheat stripe rust and lay the foundation for further in-depth analysis of its molecular mechanisms.

Key words: Wheat stripe rust, PsCON6, HIGS, Spore production gene

中图分类号:

刘贝贝, 沈煜洋, 邓菲菲, 陈江华, 李进, 李广阔, 高海峰, 李月. 小麦条锈菌产孢基因PsCON6的克隆及功能验证[J]. 华北农学报, 2025, 40(4): 185-193. doi: 10.7668/hbnxb.20195885.

LIU Beibei, SHEN Yuyang, DENG Feifei, CHEN Jianghua, LI Jin, LI Guangkuo, GAO Haifeng, LI Yue. Cloning and Functional Validation of the Spore Production Gene PsCON6 from Wheat Stripe Rust Fungus[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(4): 185-193. doi: 10.7668/hbnxb.20195885.

http://www.hbnxb.net/CN/Y2025/V40/I4/185

图/表 9

图1 PsCON6基因的克隆

Fig.1 Cloning of PsCON6 gene

图2 PsCON6蛋白保守结构域预测和蛋白跨膜结构预测 A.PsCON6的保守结构域预测;B.PsCON6的跨膜结构预测。

Fig.2 Structural domain prediction and protein transmembrane structure prediction of PsCON6 protein A.Structural domain prediction of PsCON6;B.Transmembrane structure prediction of PsCON6.

图3 PsCON6蛋白结构预测 A.PsCON6的二级结构预测;B.PsCON6的三级结构预测。

Fig.3 Prediction of PsCON6 protein structure A.Predicted secondary structure of PsCON6;B.Predicted tertiary structure of PsCON6.

图4 同物种中Con6蛋白多重序列比对及系统进化树分析 A.PsCON6的多重序列比对;B.PsCON6的系统进化树。

Fig.4 Multiple sequence alignment and phylogenetic tree analysis of Con6 protein in different species A.Multiple sequence alignment of PsCON6;B.Phylogenetic tree analysis of PsCON6.

图5 PsCON6在小麦接种条锈菌后侵染过程中的表达特征分析 US.夏孢子。试验进行3次生物学重复,同一时间点相同小写字母表示差异不显著(P>0.05),同一时间点不同小写字母之间表示差异显著(P<0.05)。图6-8同。

Fig.5 Analysis of the expression characteristics of PsCON6 in the infection process of wheat inoculated with wheat stripe rust US.Uredospore.The experiment was repeated three times,the same lowercase letters at the same time point indicate no significant difference(P>0.05),while different lowercase letters at the same time point indicate significant difference(P<0.05).The same as Fig.6-8.

图6 BSMV-HIGS技术瞬时沉默PsCON6基因后叶片产孢表型及沉默效率和生物量检测 A.PsCON6基因沉默后叶片表型(Mock.空白对照;BSMV∶PDS.阳性对照,BSMV∶γ.阴性对照);B.PsCON6基因沉默效率;C.PsCON6基因生物量检测。

Fig.6 Detection of sporulation phenotype,silencing efficiency and biomass of leaves after transient silencing of PsCON6 gene by BSMV-HIGS technique A.Phenotype of leaves after PsCON6 gene silencing(Mock.Blank control;BSMV∶PDS.Positive control,BSMV∶γ.Negative control);B.Silencing efficiency of the PsCON6 gene;C.Biomass measurement of the PsCON6 gene.

图7 BSMV-HIGS技术瞬时沉默PsCON6基因后叶片DAB染色及活性氧面积检测 A.PsCON6基因沉默后DAB染色(SV.气孔下囊;H₂O₂.活性氧);B.PsCON6基因沉默后活性氧面积检测。

Fig.7 Leaf DAB staining and active oxygen area detection after instantaneous silencing of PsCON6 gene by BSMV-HIGS technique A.DAB staining after silencing the PsCON6 gene(SV.Stomatal cavity;H₂O₂.Reactive oxygen species); B.Detection of reactive oxygen area after silencing the PsCON6 gene.

图8 BSMV-HIGS技术瞬时沉默PsCON6基因后叶片WGA染色及菌丝长度和菌丝面积检测 A.PsCON6基因沉默后WGA染色(SV.气孔下囊;IH.侵染菌丝;HMC.吸器母细胞);B.PsCON6基因沉默后菌丝长度检测;C.PsCON6基因沉默后菌丝面积检测。

Fig.8 Leaf WGA staining and mycelial length and mycelial area detection after transient silencing of PsCON6 gene by BSMV-HIGS technique A.WGA staining after PsCON6 gene silencing(SV.Substomatal vesicle;IH.Infection hyphae;HMC.Haustorial mother cell); B.Measurement of hyphal length after PsCON6 gene silencing;C.Measurement of hyphal area after PsCON6 gene silencing.

图9 PsCON6编码蛋白在烟草叶片中的亚细胞定位

Fig.9 Subcellular localization of PsCON6 encoding protein in tobacco leaves

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小麦条锈菌产孢基因PsCON6的克隆及功能验证

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