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

作　　者：杨浩蓉 贾凡 胡徐 穆蓉 刘维娜 刘昌云 王善之 孙现超[1] 马冠华[1] 陈国康[1] YANG HaoRong;JIA Fan;HU Xu;MU Rong;LIU WeiNa;LIU ChangYun;WANG ShanZhi;SUN XianChao;MA GuanHua;CHEN GuoKang(College of Plant Protection/Chongqing Key Laboratory of Plant Disease Biology,Southwest University,Chongqing 400715;Agricultural Technology Extension Center of Wanrong County,Wanrong 044200,Shanxi)

机构地区：[1]西南大学植物保护学院/植物病害生物学重庆市重点实验室,重庆400715 [2]万荣县农业技术推广中心,山西万荣044200

出　　处：《中国农业科学》2024年第19期3799-3809,共11页Scientia Agricultura Sinica

基　　金：国家重点研发计划(2023YFD1701200)。

摘　　要：【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【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,encod

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 

分 类 号：S435.654[农业科学—农业昆虫与害虫防治]