类受体激酶基因PbeCRLK2正调控梨腐烂病抗性

doi:10.3864/j.issn.0578-1752.2026.10.008

摘要/Abstract

摘要：

【目的】钙/钙调蛋白调控受体样激酶（calcium/calmodulin-regulated receptor-like kinase，CRLK）在低温胁迫和淀粉合成中发挥重要作用，但该基因在植物免疫中的功能研究较少。研究PbeCRLK2在杜梨（Pyrus betulifolia）对梨腐烂病免疫应答过程中的具体功能，为梨树抗腐烂病育种提供理论依据与基因资源。【方法】通过生物信息学和亚细胞定位，明确PbeCRLK2的基本特征。结合果实瞬时表达和悬浮细胞稳定表达体系验证PbeCRLK2的抗病功能，进一步通过活性氧（ROS）检测及免疫相关通路基因表达分析，探究其调控的下游免疫通路。对经梨腐烂病菌（Valsa pyri）代谢物处理后不同时间段的PbeCRLK2过表达细胞进行转录组分析及qRT-PCR验证，进一步研究PbeCRLK2调控梨腐烂病抗性的作用机理。【结果】PbeCRLK2是定位于细胞膜、进化保守的胞质类受体激酶，其表达可被梨腐烂病菌代谢物显著诱导。瞬时和稳定表达PbeCRLK2能显著抑制梨腐烂病菌侵染，增强杜梨对腐烂病的抗性。其中瞬时表达72 h病斑直径减少11.59%，稳定表达后病斑直径平均减少44.06%。PbeCRLK2可通过激活WRKY22表达增强模式触发免疫（PTI）响应，诱导活性氧爆发并上调RBOHD、OXI1表达，同时上调水杨酸通路关键基因ChiV的表达，协同增强病原防御能力。对转录组中DC_vs_YC、DT1_vs_YT1、DT2_vs_YT2和DT3_vs_YT3 4个组别的差异基因进行GO和KEGG富集分析发现，差异基因主要在去磷酸化、几丁质分解、脂质代谢、次生代谢产物及萜类物质合成等通路富集。qRT-PCR结果显示，所选基因的表达变化趋势与转录组数据一致，证实转录组数据准确。【结论】PbeCRLK2可能参与几丁质信号传递，进而协同激活模式触发免疫响应、活性氧爆发及水杨酸信号通路增强杜梨对腐烂病抗性，且通过调控去磷酸化相关基因动态表达维持细胞免疫稳态。

关键词: 杜梨, 梨腐烂病菌, PbeCRLK2, 抗病功能, 转录组分析

Abstract:

【Objective】CRLK (calcium/calmodulin-regulated receptor-like kinase) plays an important role in low-temperature stress and starch biosynthesis, but its function in plant immunity remains unclear. This study aimed to investigate the role of PbeCRLK2 in the immune response of Pyrus betulifolia to pear Valsa canker, and to provide theoretical basis and genetic resources for the breeding of pear trees resistant to Valsa canker.【Method】The basic characteristics of PbeCRLK2 were identified through bioinformatics and subcellular localization analysis. The role of PbeCRLK2 in disease resistance was verified using transient expression in fruits and stable expression in suspension cells. Downstream immune pathways were explored by measuring reactive oxygen species (ROS) and the expression of immune-related genes. To further investigate the mechanism by which PbeCRLK2 regulates resistance to pear Valsa canker, transcriptomic analysis and qRT-PCR verification were conducted on PbeCRLK2 overexpressing cells at different time points after treatment with metabolites of V. pyri.【Result】PbeCRLK2 was a conserved cytoplasmic receptor-like kinase localized to the cell membrane. Its expression was significantly induced by metabolites of V. pyri. Both transient and stable expression of PbeCRLK2 significantly inhibited V. pyri infection and enhanced the resistance of P. betulaefolia to pear Valsa canker. Transient expression for 72 h reduced lesion diameter by 11.59% and stable expression by 44.06% on average. PbeCRLK2 enhanced pattern-triggered immunity (PTI) responses by activating WRKY22 expression. It also induced ROS bursts and up-regulated the expression of RBOHD and OXI1. Meanwhile, the expression of the key salicylic acid pathway gene ChiV was significantly up-regulated. All of these phenomena contributed to enhanced P. betulaefolia defense capabilities. GO and KEGG enrichment analyses were conducted separately for up- and down-regulated DEGs from four groups (DC_vs_YC, DT1_vs_YT1, DT2_vs_YT2 and DT3_vs_YT3) in the transcriptome data. DEGs were mainly enriched in pathways such as protein dephosphorylation, chitin catabolic processes, lipid metabolism, secondary metabolite and terpenoid biosynthesis, etc. qRT-PCR results showed that the expression trends of the selected genes were consistent with the transcriptome data, validating the transcriptomic findings.【Conclusion】PbeCRLK2 may be involved in chitin signal transduction. PbeCRLK2 enhances the resistance of P. betulaefolia to Valsa canker through the coordinated activation of PTI responses, ROS burst, and the salicylic acid signaling pathway. At the same time, it may maintain cellular immune homeostasis by dynamically regulating the expression of dephosphorylation-related genes.

Key words: Pyrus betulaefolia, Valsa pyri, PbeCRLK2, disease resistance function, transcriptomic analysis

杨菁华, 孙娥, 刘小欢, 张盼, 戴莹葆, 王文辉, 董涵, 左存武. 类受体激酶基因PbeCRLK2正调控梨腐烂病抗性[J]. 中国农业科学, 2026, 59(10): 2165-2180.

YANG JingHua, SUN E, LIU XiaoHuan, ZHANG Pan, DAI YingBao, WANG WenHui, DONG Han, ZUO CunWu. Receptor-Like Kinase Gene PbeCRLK2 Positively Regulates the Pyrus betulaefolia Resistance to Pear Valsa Canker[J]. Scientia Agricultura Sinica, 2026, 59(10): 2165-2180.

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基因名称Gene name	正向引物Forward primer (5′-3′)	反向引物Reverse primer (5′-3′)
PbeCRLK2-pFGC5941	GAGGCGCGCCATGGTTCATCAAACTGATCTTGTTA	GAGGCGCGCCATGGTTCATCAAACTGATCTTGTTA
PbeCRLK2-GFP	GAGAACACGGGGGACGAGCTCATGGTTCATCAAA CTGATCTTGTTA	CTTGCTCACCATGGTGTCGACTTCAGTTACTCTTTC GTTCACGCTC
PbeCRLK2-qPCR	GTTATCATTGGCGTCTCTGTTGGT	TTGTGCGTATAGGTAGAGTGGTAAGA
Actin	TTCAGATACTGTTGTGGAGCCTTAC	AGTAACTCCAGACATTGTTGCAGAG
LOX1	GCTTATGTGGCTGTAAATGACTCTG	GAGGATGCAGAAGTTTGTAAATTGG
ChiV	CTACAGCATAAACTACCCCTTCCAG	CGTTCCTATACCGGATCCATTAGC
RBOHD	TACGTGGAGATCACTCTCGACATC	CCGCTTCAGCTCCTGAGAGAC
OXI1	CAACTTGGAAAACTCCGAGAAGTC	TTGTCAGTATCCGATAAAACGTCTC
WRKY22	CATATCCAAGGGGATATTACAGATG	GTGACTATAAAAATATTCGGGTCGG

样本 Sample	原始数据 Raw data	过滤后数据 Clean data	过滤后数据比率 Clean data ratio (%)	Q20 (%)	Q30 (%)	GC (%)
YC-1	41866810	41866810	100	98.13	96.73	46.75
YC-2	43720318	43720318	100	98.36	96.97	46.65
YC-3	37260460	37260460	100	98.11	96.70	46.84
YT1-1	40110802	40110802	100	98.06	96.63	46.57
YT1-2	36265408	36265408	100	98.17	96.81	46.52
YT1-3	36308808	36308808	100	98.07	96.61	46.62
YT2-1	45657690	45657690	100	98.47	97.08	46.31
YT2-2	39679760	39679760	100	98.25	96.92	46.35
YT2-3	38180810	38180810	100	98.16	96.78	46.15
YT3-1	41359618	41359618	100	98.30	97.01	46.03
YT3-2	42150220	42150216	100	98.40	96.97	46.09
YT3-3	41439948	41439948	100	98.37	97.12	46.11