甘蓝型油菜MYB4基因RNA干扰载体构建  

Construction of RNAi Vector of Brassica napus MYB4 Gene

作　　者：王玉明[1] 曹廷[1] 冯瑜[1] 柴友荣[1] 

机构地区：[1]西南大学农学与生物科技学院、农业部生物技术与作物品质改良重点实验室、重庆市作物品质改良重点实验室、重庆市油菜工程技术研究中心,重庆北碚400716

出　　处：《作物杂志》2010年第2期23-27,共5页Crops

基　　金：重庆市科技攻关项目(CSTC2009AB1030);国家“863”计划(2006AA10Z110)

摘　　要：拟南芥(Arabidopsis thaliana)MYB4(AtMYB4)编码负调控转录因子,通过对苯丙烷途径中关键酶C4H(肉桂酸4-羟化酶)基因的抑制来调控重要次生代谢物质芥子酸酯的生物合成,在防御紫外线中起重要作用。同为十字花科的芸薹属(Brassica)有多种重要的油料、蔬菜和园林作物,如甘蓝型油菜(B.napus)、白菜(B.rapa)、甘蓝(B.oleracea)和芥菜(B.juncea)等。本研究克隆了甘蓝型油菜MYB4基因家族的RNA干扰片段BMYB4I,将其反义片段、正义片段采用NcoI+AatII和BamHI+XbaI分别插入到改进型植物RNA干扰基础载体pFGC5941M的启动子与间隔区、间隔区与终止子之间,形成重组载体pF-GC5941M-BMYB4I(简称为pBMYB4I)。经复合PCR鉴定表明,载体构建成功,并转化根癌农杆菌获得工程菌株。为揭示芸薹属MYB4基因调控芥子酸酯等苯丙烷物质合成的分子机理以及探索抗紫外线分子育种奠定了基础。Arabidopsis thaliana MYB4（AtMYB4）encodes a negative-regulating transcription factor,controls the biosynthesis of important secondary metabolites sinapate esters,and plays an important role in defensing ultraviolet,by repressing the transcription of key enzyme gene C4H（cinnamate 4-hydroxylase）.Also from Brassicaceae,the genus Brassica contains many important oilseed,vegetable and ornamental crops,such as B.napus,B.rapa,B.oleracea and B.juncea from the U-triangle.In this study,an RNAi fragment BMYB4I targeted to Brassica napus MYB4 gene family was cloned,and its antisense and sense fragments were inserted to the promoter-spacer and spacer-terminator cloning sites using NcoI＋AatII and BamHI＋XbaI double digestions respectively.The resulting recombinant vector was pFGC5941M-BMYB4I（simplified as pBMYB4I）,which was verified by multiple PCR checking and successfully transformed into Agrobacterium to form engineering strain,laying a basis for clarifying the molecular mechanism of Brassica MYB4 gene family in regulating the accumulation of sinapate esters and other phenylpropanoids and for molecular breeding of ultraviolet tolerance.

关 键 词：芸薹属 MYB4 RNAI 载体构建 紫外线 

分 类 号：S565.4[农业科学—作物学]