脂氧合酶基因CsLOX2-1调节甜橙抗病的功能

doi:10.3864/j.issn.0578-1752.2026.14.006

摘要/Abstract

摘要：

【目的】基于甜橙（Citrus sinensis）与砂皮病菌（Diaporthe citri）互作转录组数据，筛选获得一个显著上调的脂氧合酶基因CsLOX2-1，系统解析其表达特征与抗病功能，为柑橘抗病育种提供候选基因资源。【方法】利用RT-qPCR技术检测CsLOX2-1在甜橙不同组织（根、茎、叶、果实、种子）及多种胁迫条件（4、40 ℃、100 mmol·L^-1 NaCl高盐处理、机械损伤、柑橘溃疡病菌（Xanthomonas citri subsp. citri，Xcc）与砂皮病菌侵染）下的表达模式。通过生物信息学分析CsLOX2-1的结构域以及与其他物种LOX基因的进化关系。利用农杆菌介导的瞬时表达技术，在甜橙叶片中瞬时过表达CsLOX2-1，接种溃疡病菌后评估其对溃疡病的抗性，并检测茉莉酸（JA）含量变化。此外，构建拟南芥异源表达植株，应用模式菌株丁香假单胞杆菌番茄致病变种（Pseudomonas syringae pv. tomato，Pst DC3000）和灰霉病菌（Botrytis cinerea）分析其对细菌和真菌的抗性，并通过RT-qPCR检测防御相关基因的表达水平。【结果】甜橙CsLOX2-1的CDS全长为2 703 bp，编码900个氨基酸，该蛋白含有一个典型的Lipoxygenase结构域和一个PLAT/LH2结构域，无跨膜结构域。该基因与楝中同源基因的相似性高达99%，定位于叶绿体。表达模式分析表明，CsLOX2-1在甜橙不同组织中呈现特异性表达，叶片中相对表达量最高；多种胁迫下，其对生物胁迫的响应尤为显著，柑橘溃疡病菌、砂皮病菌侵染48 h时各上调17.64、257.10倍。瞬时过表达试验显示，在甜橙叶片中过表达CsLOX2-1可显著促进茉莉酸的合成，同时显著增强对溃疡病的抗性。在拟南芥中异源过表达CsLOX2-1后，多个防御相关基因（AtJAR1、AtMYC2、AtPDF1.2、AtPR1等）的表达水平显著上调，同时对Pst DC3000及灰霉病菌的抗性也显著提升。【结论】CsLOX2-1受病原菌侵染诱导表达，通过调控茉莉酸信号通路参与柑橘抗病。研究结果可为柑橘抗病育种提供重要的基因资源。

关键词: 甜橙, 脂氧合酶, 基因功能, 瞬时表达, 遗传转化

Abstract:

【Objective】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.【Method】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.【Result】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.【Conclusion】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.

Key words: Citrus sinensis, lipoxygenase, gene function, transient expression, genetic transformation

罗长威, 邓杰夫, 王冰, 易图永, 宋娜. 脂氧合酶基因CsLOX2-1调节甜橙抗病的功能[J]. 中国农业科学, 2026, 59(14): 3070-3081.

LUO ChangWei, DENG JieFu, WANG Bing, YI TuYong, SONG Na. The Disease Resistance Function Mediated by the Lipoxygenase Gene LOX2-1 in Citrus sinensis[J]. Scientia Agricultura Sinica, 2026, 59(14): 3070-3081.

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)	引物名称 Primer name	引物序列 Primer sequence (5′-3′)
CsLOX2-1-F	ATGTTGAAGCCACAGGTTCATCAAC	qPCR-AtPDF1.2-F	GCTTCCATCATCACCCTTATC
CsLOX2-1-R	GATAGAGATGCTGTACGGAACTCC	qPCR-AtPDF1.2-R	GTCCCACTTGGCTTCTCG
RT-qPCR-CsLOX2-1-F	ACCTTCTTGGCACTCTACT	qPCR-AtPR1-F	TACGCAGAACAACTAAGAGGC
RT-qPCR-CsLOX2-1-R	TGTCATAGGCAACAGAGC	qPCR-AtPR1-R	AGACAAGTCACCGCTACCC
CsLOX2-1-EGFP-F	CGGGGGACGAGCTCGATGTTGAAGCCACAGGTTCATCAAC	qPCR-AtPR2-F	CTCAAGGAAGGTTCAGGGAT
CsLOX2-1-EGFP-R	ACGAGATCTGGTCGAGATAGAGATGCTGTACGGAACTCC	qPCR-AtPR2-R	GAGATTCACGAGCAAGGGA
qPCR-CsCOX-F	GTATGCCACGTCGCATTCCAGA	qPCR-AtPR3-F	GACACCGCCACGAGGAAG
qPCR-CsCOX-R	GCCAAAACTGCTAAGGGCATTC	qPCR-AtPR3-R	CGTAGTAGCGTTTGCCAGAT
1300-EGFP-F	TGAGACTTTTCAACAAAGGGTAATACCG	qPCR-AtPR5-F	TGACTCCAGGTGCTTCCC
1300-EGFP-R	TGAACAGCTCCTCGCCCTTG	qPCR-AtPR5-R	CGCCGCCGTTACATCTTAG
qPCR-AtActin7-F	CCATTCAGGCCGTTCTTTC	qPCR-AtACO-F	GGGGTTCTACTGCGTTCC
qPCR-AtActin7-R	CGTTCTGCGGTAGTGGTGA	qPCR-AtACO-R	CATCCAGCAACTCCCAAAC
qPCR-AtJAR1-F	CGGCTTGGAGATGTGGTA	qPCR-AtCTR1-F	CGGACGAGTCTGCTTTACG
qPCR-AtJAR1-R	CGATGAATGCTCGGTCTAA	qPCR-AtCTR1-R	TTGAAGGGATGCGACCAC
qPCR-AtMYC2-F	GCAACCGTCGTATGATTTCT	qPCR-AtWRKY26-F	TCCTCCCTTGTTCCTACCA
qPCR-AtMYC2-R	ATTCCGTATCCGTCACCTC	qPCR-AtWRKY26-R	TTCCTTTGACTTGCTTCTGC