The Disease Resistance Function Mediated by the Lipoxygenase Gene LOX2-1 in Citrus sinensis

Based on the transcriptome data of Citrus sinensis interaction with Diaporthe citri, a significantly up-regulated lipoxygenase gene was screened and named CsLOX2-1. The aim of this study is to systematically analyze its expression characteristics and disease resistance function, and to provide candidate gene resources for citrus disease resistance breeding.

The expression pattern of CsLOX2-1 in different tissues (roots, stems, leaves, fruits, seeds) of C. sinensis and under various stress conditions (4, 40 ℃, 100 mmol·L^-1 NaCl, mechanical damage, infection with Xanthomonas citri subsp. citri (Xcc) and D. citri) was detected using RT-qPCR. Bioinformatics analysis was performed to investigate the domain structure of CsLOX2-1 and its evolutionary relationship with LOX genes from other species. Agrobacterium-mediated transient expression technology was used to overexpress CsLOX2-1 in C. sinensis leaves, followed by evaluation of its resistance to citrus canker after Xcc inoculation and detection of jasmonic acid (JA) content changes. Additionally, Arabidopsis thaliana heterologous expression lines were constructed, and their resistances to the model strains Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) and Botrytis cinerea were analyzed, and the expression levels of defense-related genes were detected by RT-qPCR.

The full-length CDS of CsLOX2-1 is 2 703 bp, encoding 900 amino acids. The protein contains a typical Lipoxygenase domain and a PLAT/LH2 domain, without transmembrane domain. This gene shares 99% similarity with its homolog in Melia azedarach and is localized to the chloroplast. Expression pattern analysis revealed that CsLOX2-1 exhibited tissue-specific expression in C. sinensis, with the highest relative expression in leaves. Under multiple stresses, the response to biotic stress was particularly significant, which was up-regulated by 17.64 and 257.10 times at 48 h after infection with Xcc and D. citri, respectively. Transient overexpression experiments showed that overexpressing CsLOX2-1 in C. sinensis leaves significantly promoted JA synthesis and enhanced resistance to citrus canker. After heterologous overexpression of CsLOX2-1 in A. thaliana, the expression levels of multiple defense-related genes (AtJAR1, AtMYC2, AtPDF1.2, AtPR1, etc.) were significantly up-regulated, accompanied by a marked enhancement in resistance to Pst DC3000 and B. cinerea.

CsLOX2-1 is induced by pathogen infection and participates in citrus disease resistance by regulating the JA signaling pathway. The research results can provide important gene resources for citrus disease resistance breeding.