核桃JrPR1和JrGLU鉴定及抗炭疽病功能分析

doi:10.16420/j.issn.0513-353x.2024-0558

摘要/Abstract

摘要：

病程相关基因（Pathogenesis-related gene，PR）和β-1,3-葡聚糖酶（β-1,3-glucanase）基因在植物抵御病害中具有重要作用。以炭疽病抗性存在明显差异的‘鲁康8号’和‘香玲’核桃为试材体外接种胶孢炭疽菌，10 d后‘香玲’青皮病斑明显增大，而‘鲁康8号’无明显变化。转录组分析发现二者青皮的差异基因富集在植物—病原互作、MAPK信号途径和植物激素信号转导等抗病相关途径，并初步筛选到JrPR1和JrPR5、JrGLU和JrGLU5在抗病品种‘鲁康8号’中高表达。组织特异性分析表明JrPR5和JrGLU5分别在芽和叶片中高表达，JrPR1和JrGLU在果实中高表达，可作为调控果实炭疽病抗性候选基因。烟草叶片瞬时表达发现JrPR1定位于细胞壁和细胞间隙，JrGLU定位于细胞壁，并分析了它们编码蛋白的理化性质，二级和三级结构。核桃果实瞬时表达发现JrPR1和JrGLU均能明显抑制胶孢炭疽菌生长，提高果实炭疽病抗性。

关键词: 核桃, 病程相关基因, β-1,3-葡聚糖酶, 炭疽病

Abstract:

Pathogenesis-related genes and β-1,3-glucanase genes play important roles in plant disease resistance.‘Lukang 8’and‘Xiangling’walnut cultivars，which exhibit significant differences in anthracnose resistance，were used as experimental materials. 10 days after in vitro inoculation with Colletotrichum gloeosporioides，the husk lesion on‘Xiangling’significantly enlarged，while‘Lukang 8’showed no obvious changes. Transcriptome analysis revealed that the differentially expressed genes in the husks of the two cultivars were enriched in disease resistance-related pathways such as plant-pathogen interaction，MAPK signaling pathway，and plant hormone signal transduction. The preliminarily identified that JrPR1，JrPR5，JrGLU and JrGLU5 are highly expressed in the disease-resistant cultivar‘Lukang 8’. The tissue-specific analysis showed that JrPR5 and JrGLU5 are highly expressed in buds and leaves，respectively，while JrPR1 and JrGLU are highly expressed in fruits，suggesting they are candidate genes for regulating fruit anthracnose resistance. Transient expression in tobacco leaves revealed that JrPR1 is localized to the cell wall and intercellular spaces，while JrGLU is localized to the cell wall. Additionally，the physicochemical properties，secondary，and tertiary structures of their encoded proteins were analyzed. Transient expression in walnut fruits revealed that both JrPR1 and JrGLU can significantly inhibit the growth of C. gloeosporioides and enhance anthracnose resistance in the fruits.

Key words: walnut, pathogenesis-related gene, β-1,3-glucanase, anthracnose

许海峰, 王贵芳, 相昆, 沈广宁, 张美勇, 陈新. 核桃JrPR1和JrGLU鉴定及抗炭疽病功能分析[J]. 园艺学报, 2025, 52(7): 1696-1706.

XU Haifeng, WANG Guifang, XIANG Kun, SHEN Guangning, ZHANG Meiyong, and CHEN Xin. Identification and Functional Analysis of Walnut JrPR1 and JrGLU in Relation to Anthracnose Resistance[J]. Acta Horticulturae Sinica, 2025, 52(7): 1696-1706.

图/表 8

图1 ‘鲁康8号’和‘香玲’核桃炭疽病抗性分析

Fig. 1 Analysis of anthracnose resistance in‘Lukang 8’and‘Xiangling’walnuts

图2 ‘鲁康8号’和‘香玲’核桃接种胶孢炭疽菌后基因富集分析 A：主成分分析（PCA）；B：KEGG分析。T：鲁康8号；X：香玲。T0和T10、X0和X10表示胶孢炭疽菌侵染0和10 d。下同

Fig. 2 Gene enrichment analysis of‘Lukang 8’and‘Xiangling’walnuts after inoculation with Colletotrichum gloeosporioides A：Principal component analysis（PCA）；B：KEGG analysis. T：Lukang 8；X：Xiangling. T0 and T10，X0 and X10 represent the 0 days and 10 days post-infection with Colletotrichum gloeosporioides，respectively. The same below

图3 接种胶孢炭疽菌后10 d‘鲁康8号’和‘香玲’核桃青皮PR和GLU基因表达热图颜色从蓝到红表示基因表达水平越高

Fig. 3 Heatmap of PR and GLU genes between‘Lukang 8’and‘Xiangling’walnut’s husks on day 10 post-inoculation with Colletotrichum gloeosporioides Colors ranging from blue to red indicate higher levels of gene expression

图4 PR和GLU组织特异性表达不同小写字母表示组织间差异显著，P < 0.01

Fig. 4 Tissue-specific expression of PR and GLU genes Different lowercase letters indicate significant differences between tissues，P < 0.01

图5 PR1和GLU烟草叶片亚细胞定位分析

Fig. 5 Subcellular localization analysis of PR1 and GLU in tobacco leaves

表1 PR1和GLU蛋白理化性质、二级结构

Table 1 Physicochemical properties and secondary structure of PR1 and GLU proteins

基因名 Gene name	染色体定位 Chromosomal location	分子量/kD Molecular weight	等电点 Isoelectric point	平均亲水性 Average hydrophilicity	不稳定指数 Instability index	α螺旋/% Alpha helix	延伸链/% Extended strand	β转角/% Beta turn	无规则卷曲/% Random coil
PR1	NC_049913.1	22.10	9.51	-0.06	31.99	40.40	17.68	3.03	38.89
GLU	NC_049905.1	37.80	4.80	-0.10	34.49	35.63	20.11	5.75	38.51

图6 PR1和GLU蛋白结构分析 A：PR1和GLU二级结构排列；B：PR1和GLU三级结构模型

Fig. 6 Structural analysis of PR1 and GLU proteins A：Secondary structure arrangement of PR1 and GLU proteins；B：Tertiary structure models of PR1 and GLU proteins

图7 瞬时表达PR1和GLU并接种胶孢炭疽菌6 d后核桃果实抗性分析 pIR：IL60-1 + IL60-2；pIR-PR1：IL60-1 + PR1-IL60-2；pIR-GLU：IL60-1+ GLU-IL60-2。不同小写字母表示差异显著，P < 0.01

Fig. 7 Resistance analysis of walnut fruits after transient expression of PR1 and GLU and inoculation with Colletotrichum gloeosporioides for 6 d pIR：IL60-1 + IL60-2；pIR-PR1：IL60-1 + PR1-IL60-2；pIR-GLU：IL60-1+ GLU-IL60-2. Different lowercase letters indicate significant differences，P < 0.01

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