大麦根部快速稳定转化体系的建立  

Establishment of rapid and stable root transformation system in barley

作　　者：李亮[1] 王晓阳[1] 主朋月 穆迪[1] 武洪庆[1] 齐树亭[1] Liang Li Xiaoyang Wang Pengyue Zhu Di Mu Hongqing Wu Shuting Qi(School of Marine Science and Engineering, Hebei University of Technology, Tianjin 300130, China)

机构地区：[1]河北工业大学海洋科学与工程学院,天津300130

出　　处：《生物技术》2016年第5期487-494,510,共9页Biotechnology

基　　金：河北省自然科学基金项目(No.C2014202256);天津市自然科学基金项目(No.13JCYBJC37600);河北省人力资源与社会保障厅项目(No.C201400308);河北省科技支撑计划项目(No.12273301)

摘　　要：[目的]为了解决大麦根组织与根际微生物(有益菌或致病菌)互作分析系统的缺失,建立了大麦根部快速稳定遗传转化系统。[方法]显微镜下通过分离大麦未成熟幼胚并剥离胚轴,对大麦幼胚实施含有外源基因GFP、Hv EXPANSINB1-RNAi、BAX INHIBITOR-1以及GFP-ER的农杆菌转化,将大麦幼胚分别转入愈伤培养基和生根培养基进行根组织的诱导。[结果]大量的根可以在6 w时间内被成功诱导。利用该体系:实现GFP在根中表达;转有Hv EXPANSINB1-RNAi基因的根毛数量显著减少;经BAX INHIBITOR-1基因转化的根组织减少了病原菌Fusarium graminearum的定殖;成功地检测到了GFP-ER基因在内质网及细胞核周围的定殖。[结论]该体系可成功应用于外源基因的表达、沉默,大麦根部功能基因与病原菌的互作以及蛋白的亚细胞定位研究。[ Objective] A major drawback in the characterization of barley genes supporting or restricting root colonization by mutualistic or pathogenic fungi is the lack of a highly flexible and fast barley root transformation system. Such a transformation system has been established for solving the above problem. [ Methods] The immature embryo was isolated under the microsco- py. The scutellum was transformed with the Green Fluorescent Protein （GFP）, BAX inhibitor（ B1- 1 ）, HvEXPANSINB1 - RNAi and GFP - ER genes via Agrobacterium in dark. Afterwards, the scutellum was transferred into barley callus induction medium and root induction medium. [ Results] Large scale production of transgenic barley roots was obtained within 6 weeks. GFP expression and HvEPXANSINB1 silencing phenotype was observed in the transgenic roots,in addition, the root transformed with BAX INHIBITOR - 1 reduced the Fusarium graminearum colonization. Finally ,the GFP - ER protein locolization was observed in the endoplasmic reticulum and the nuclear periphery. [ Conclusion ] The rapid and stable root transformation system could be used for gene expression, silencing, protein subcellular localization as well as the analysis of barley root - microbe interactions.

关 键 词：大麦 快速 稳定 根部转化 病原菌与大麦互作 

分 类 号：Q819[生物学—生物工程]