油菜BnJAZ7通过调控抗氧化途径促进核盘菌侵染

BnJAZ7 Promotes Sclerotinia sclerotiorum Infection by Affecting the Antioxidant Pathway in Brassica napus

【目的】由核盘菌(Sclerotinia sclerotiorum)引起的油菜菌核病是危害油菜生产的第一大病害。通过分子克隆获得甘蓝型油菜BnJAZ7,利用生物信息学、细胞生物学以及分子生物学手段,明确甘蓝型油菜BnJAZ7的表达特征及其在核盘菌侵染过程中的作用,为油菜抗菌核病分子育种打下理论基础。【方法】利用分子克隆技术扩增BnJAZ7全长,并分析其生物信息学特征,利用MEGA X构建BnJAZ7的亲缘关系树。利用实时荧光定量PCR技术测定BnJAZ7在油菜中的组织表达以及核盘菌侵染过程中的表达特征;构建BnJAZ7与GFP的融合表达载体,在激光共聚焦显微镜下观察融合蛋白的亚细胞定位。在本氏烟叶片中瞬时表达融合蛋白,并接种核盘菌,分析BnJAZ7的表达对核盘菌侵染的影响。构建BnJAZ7-OE过表达转基因油菜,在其T2代叶片上接种核盘菌,观察其对核盘菌侵染的影响。利用生化技术检测核盘菌侵染BnJAZ7-OE后抗氧化酶SOD、POD、CAT以及PAL活性;通过实时荧光定量PCR技术分析核盘菌侵染BnJAZ7-OE油菜后相关基因BnSOD、BnPOD、BnPAL和BnCAT的转录水平。【结果】BnJAZ7全长801 bp,编码266个氨基酸,由TIFY和CCT_2两个结构域组成,分子式为C_(1244)H_(2000)N_(358)O_(384)S_(13),等电点为9.57,理论分子量为28.53 kDa,不具跨膜结构域。BnJAZ7与油菜TIFY 7的亲缘关系最近,在油菜茎中表达量最高,并且核盘菌侵染后6、12、24、48 h其表达量持续上升。亚细胞定位显示eGFP:BnJAZ7定位于细胞膜和细胞核。在本氏烟叶片中异源瞬时表达eGFP:BnJAZ7促进核盘菌侵染;BnJAZ7过表达的BnJAZ7-OE转基因油菜也促进核盘菌侵染。核盘菌在BnJAZ7-OE植株中侵染后显著降低了BnPOD、BnSOD和BnPAL的转录水平,显著增加了BnCAT的转录水平,从而降低POD、SOD和PAL活性,增加CAT活性。【结论】甘蓝型油菜BnJAZ7的表达受到核盘菌侵染诱导,且BnJAZ7过表达显著加快了核盘菌的侵染速度。核盘菌通过诱导BnJAZ7上升后降低抗氧化酶编码基因BnPOD、BnSOD和BnPAL转录水平、增加BnCAT转录水平,从而调控抗氧化酶活性来进一步加快侵染。

【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 C_(1244)H_(2000)N_(358)O_(384)S_(13),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.