北京林业大学学报2026，Vol.48Issue(6)：152-163,12.DOI:10.12171/j.1000-1522.20260175
油松响应松材线虫侵染的m6A甲基化修饰特征分析及关键基因功能验证
Characteristics of m6A methylation modification of Pinus tabuliformis in response to Bursaphelenchus xylophilus infection and functional verification of key genes
摘要
Abstract
[Objective]Pine wilt disease has posed a serious threat to the Pinus tabuliformis forest ecosystem in China.Currently,the epitranscriptomic regulatory mechanisms of conifers in response to pathogen infection remain unclear.This study aimed to investigate the dynamic changes of the m6A methylation landscape in P.tabuliformis upon B.xylophilus infection,screen and validate key m6A-regulated R genes,elucidate the molecular mechanism of m6A-mediated R gene regulation,and construct a"methylase-m6A-R gene-resistance"regulatory model,thereby providing theoretical foundations and genetic resources for molecular breeding of disease-resistant conifers.[Method]Three-year-old P.tabuliformis seedlings were used as materials.Fourteen days after inoculation with B.xylophilus,the stem segments were subjected to whole-transcriptome and m6A modification profiling using Oxford Nanopore direct RNA sequencing.Multi-omics integrative analysis was performed to screen the key candidate gene PtLRR-XI1,which was then heterologously transformed into Arabidopsis thaliana via Agrobacterium-mediated transformation.Its disease resistance function was validated by assessing infection incidence and antioxidant physiological indicators.The methylation modification was validated by co-expression in tobacco.[Result]After B.xylophilus infection,a total of 4 527 differentially expressed genes were identified in the stem segments of P.tabuliformis,with downregulated expression of methyltransferase genes and upregulated expression of demethylase genes.A total of 62 787 differentially methylated sites were identified at the genome-wide level,predominantly enriched in the 3 'UTR regions,exhibiting a conserved"RRACH"motif.Integrative analysis identified 268 transcripts with simultaneous significant changes in expression and methylation,which were enriched in the plant-pathogen interaction and MAPK signaling pathways.Among these,PtLRR-XI1,a gene belonging to the LRR-RLK family,was identified.Its promoter contains multiple hormone-and stress-responsive elements,and it is highly conserved in conifers.Heterologous overexpression of this gene reduced the incidence of pine wilt disease in transgenic A.thaliana from 72.9%in the wild type to 25.0%-39.6%.Furthermore,after site-directed mutagenesis of two differential m6A modification sites in the 3 'UTR of PtLRR-XI1,transient transformation in Nicotiana benthamiana leaves showed that the GFP fluorescence intensity of the mutated sequence was significantly weaker than that of the unmutated control,indicating that the expression of PtLRR-XI1 is regulated by m6A modification.[Conclusion]B.xylophilus infection induces reprogramming of the m6A methylation modification system in P.tabuliformis.PtLRR-XI1 is a key resistance gene regulated by this modification,and its overexpression enhances the resistance of A.thaliana to B.xylophilus.The m6A modification sites in the 3'UTR region of this gene positively regulate its expression.This study offers a potential gene target for molecular breeding of disease resistance in P.tabuliformis.关键词
油松/松材线虫/m6A甲基化/R基因/抗病分子育种/基因表达Key words
Pinus tabuliformis/Bursaphelenchus xylophilus/m6A methylation/R gene/molecular breeding for disease resistance/gene expression分类
农业科技引用本文复制引用
桑悦,栗宇鹏,王美玲,焦志远,李伟..油松响应松材线虫侵染的m6A甲基化修饰特征分析及关键基因功能验证[J].北京林业大学学报,2026,48(6):152-163,12.基金项目
科技创新2030重大项目课题(2022ZD0401605). （2022ZD0401605）
