关键词: 甘蓝型油菜, BnJAZ7, 核盘菌, 基因表达, 油菜菌核病, 抗氧化酶

Abstract:

【Objective】Rapeseed sclerotiniose caused by Sclerotinia sclerotiorum, is the first major disease affecting rapeseed production. The objective of this study is to obtain BnJAZ7 from Brassica napus via molecular cloning, and to clarify the expression characteristics of BnJAZ7 and its role in the process of S. sclerotiorum infection by means of bioinformatics, cell biology and molecular biology, so as to lay a theoretical foundation for molecular breeding of sclerotiniose resistance in rapeseed.【Method】The full-length of BnJAZ7 was amplified using molecular cloning techniques, and the protein characteristics encoded by BnJAZ7 were analyzed through bioinformatics. The genetic relationship tree of BnJAZ7 was constructed using MEGA X. Real-time fluorescence quantitative PCR technology was employed to analyze the tissue-specific expression of BnJAZ7 in rapeseed, as well as its expression during S. sclerotiorum infection. A fusion expression vector of BnJAZ7 and GFP was generated and transiently transformed via Agrobacterium-mediated delivery into Nicotiana benthamiana leaf cells. Subsequently, the subcellular localization of the fusion protein was observed under a confocal laser microscope. The fusion protein was transiently expressed in N. benthamiana leaves and inoculated with S. sclerotiorum to assess the impact of BnJAZ7 expression on S. sclerotiorum infection. The BnJAZ7-OE overexpression transgenic rapeseed lines were developed. Following the generation of transgenic plants with high BnJAZ7 expression, the leaves of the T2 generation were inoculated with S. sclerotiorum to evaluate its effect on S. sclerotiorum infection. Biochemical techniques were utilized to measure the activities of antioxidant enzymes SOD, POD, CAT and PAL after S. sclerotiorum infection in BnJAZ7-OE rapeseed. Furthermore, real-time fluorescence quantitative PCR technology was applied to analyze the expression of antioxidant pathway-related genes BnSOD, BnPOD, BnPAL and BnCAT after S. sclerotiorum infection in BnJAZ7-OE rapeseed.【Result】BnJAZ7 spans 801 bp, encoding a protein of 266 aa, characterized by two structural domains, TIFY and CCT_2. Its molecular formula is C₁₂₄₄H₂₀₀₀N₃₅₈O₃₈₄S₁₃, with an isoelectric point of 9.57 and a theoretical molecular weight of 28.53 kDa. Notably, it lacks a transmembrane domain. Phylogenetic analysis revealed the closest genetic affinity of BnJAZ7 with B. napus TIFY 7. Expression profiling indicated a descending trend of BnJAZ7 expression in rapeseed tissues, from stems to roots, leaves, and flowers. However, after infection with S. sclerotiorum, BnJAZ7 expression showed a sequential increase at 6, 12, 24, and 48 hours post-infection. Subcellular localization studies demonstrated that eGFP:BnJAZ7 localized within the cell membrane and nucleus. Heterologous transient expression of eGFP:BnJAZ7 in N. benthamiana leaves enhanced the invasion of S. sclerotiorum. Additionally, overexpression of BnJAZ7 in transgenic rapeseed (BnJAZ7-OE) promoted S. sclerotiorum infection. After S. sclerotiorum infection in BnJAZ7-OE plants, the transcription levels of BnPOD, BnSOD and BnPAL were significantly reduced, and the transcription level of BnCAT was significantly increased, thereby reducing POD, SOD and PAL activities and increasing CAT activity.【Conclusion】The expression of BnJAZ7 in B. napus is stimulated by S. sclerotiorum infection. Moreover, the overexpression of BnJAZ7 significantly accelerates the infection rate of S. sclerotiorum. The pathogen further expedites infection by upregulating BnJAZ7 expression, subsequently diminishing the transcription levels of BnPOD, BnSOD and BnPAL, and increasing the transcription level of BnCAT, thereby regulating the activity of antioxidant enzymes to further accelerate the infection.

Key words: Brassica napus, BnJAZ7, Sclerotinia sclerotiorum, gene expression, rapeseed sclerotiniose, antioxidant enzyme