大豆NADPH氧化酶基因GmRbohL在共生结瘤过程中的功能鉴定  

Functional Identification of Soybean NADPH Oxidase Gene GmRbohL in the Nodulation Process

作　　者：柯丹霞 冯爽 胡艺涵 许丹 王环 KE Danxia;FENG Shuang;HU Yihan;XU Dan;WANG Huan(College of Life Sciences,Xinyang Normal University,Institute for Conservation and Utilization of Agro-bioresources in Dabie Mountains,Xinyang 464000,China)

机构地区：[1]信阳师范大学生命科学学院,大别山农业生物资源保护与利用研究院,河南信阳464000

出　　处：《华北农学报》2023年第5期29-38,共10页Acta Agriculturae Boreali-Sinica

基　　金：国家自然科学基金项目(U1904102);河南省高等学校青年骨干教师培养计划项目(2020GGJS152);信阳师范大学“南湖学者奖励计划”青年项目;信阳师范大学大学生科研基金项目。

摘　　要：植物NADPH氧化酶Rbohs是活性氧(ROS)的主要来源,参与植物的生长、发育、抗逆和植物-微生物的互作等多种生理过程。为探索Rbohs在共生固氮中的功能和作用机制,克隆了1个大豆Rbohs基因家族成员GmRbohL,利用分子生物学、细胞生物学和遗传学等方法,分别对基因的表达模式、蛋白质的亚细胞定位及基因的功能进行了研究。结果显示:GmRbohL基因受根瘤菌诱导,在大豆根和根瘤中特异性高表达。亚细胞定位分析发现,该基因编码蛋白GmRbohL是一种膜蛋白。构建了GmRbohL的植物基因沉默(RNAi)载体,利用发根农杆菌K599介导的大豆毛根转化法,得到转基因毛状根。GmRbohL的基因沉默导致转基因毛状根的根瘤数量明显减少,ROS的产生也受到了抑制。根瘤器官发生阶段根瘤菌的侵染事件明显减少,结瘤标志基因的表达水平也随GmRbohL表达量的降低而降低。根瘤组织切片显示,GmRbohL的基因沉默显著减少了根瘤侵染区共生体的数量,根瘤的固氮酶活性也相应降低。以上数据表明,GmRbohL的基因沉默降低了ROS的产生水平,显著抑制了大豆的共生结瘤过程。推测GmRbohL可能在大豆根瘤的器官发生以及固氮功能调控方面发挥重要的正调控作用。The plant NADPH oxidase Rbohs(Respiratory burst oxidase homologs)is the main source of reactive oxygen species(ROS),which participate in various physiological processes such as plant growth,development,stress resistance and plant-microorganism interaction.In order to explore the function and mechanism of Rbohs in symbiotic nitrogen fixation,GmRbohL,a member of soybean Rbohs gene family,was cloned in this study.The gene expression pattern,protein subcellular localization and gene function were studied by molecular biology,cell biology and genetics,respectively.The results revealed that:GmRbohL gene was induced by rhizobia and expressed specifically in soybean roots and nodules.Subcellular localization analysis indicated that the gene-encoded protein GmRbohL was a membrane protein.The plant gene silencing(RNAi)vector of GmRbohL was constructed,and the transgenic hairy roots were obtained by the transformation of soybean hairy root mediated by Agrobacterium rhizogenes K599.Gene silencing of GmRbohL resulted in a significant reduction in the number of nodules of transgenic hairy roots,and the production of ROS was also inhibited.Gene silencing of GmRbohL reduced the infection of rhizobia at the stage of root nodule organogenesis,and the expression level of nodulation marker genes also decreased with the decrease of GmRbohL expression.The root nodule tissue sections showed that gene silencing of GmRbohL significantly reduced the number of symbionts in the infected area of root nodules,and the nitrogenase activity of root nodules also decreased accordingly.The above data indicated that gene silencing of GmRbohL significantly inhibits the symbiotic nodulation process of soybean by reducing the production level of ROS.It is speculated that GmRbohL may play an important positive regulatory role in organogenesis of soybean nodules and regulation of nitrogen fixation function.

关 键 词：大豆 Rbohs 活性氧 RNAI 根瘤形成 共生结瘤 

分 类 号：S565.03[农业科学—作物学] Q78[生物学—分子生物学]