芍药切花ACS和ETR1基因的克隆、时空表达及功能分析

项目名称： 芍药切花ACS和ETR1基因的克隆、时空表达及功能分析

项目编号： No.U1204323

项目类型： 联合基金项目

立项/批准年度： 2013

项目学科： 植物保护学、园艺学与植物营养学

项目作者： 范丙友

作者单位： 河南科技大学

项目金额： 30万元

中文摘要： 芍药切花的观赏价值极高，但保鲜技术及衰老机制的研究相对滞后，严重影响了其产业化进程。本研究以已鉴定的类似跃变型及类似末期上升型两种乙烯代谢类型的芍药切花为研究材料，基于RACE技术分离乙烯生物合成关键酶ACS和乙烯受体ETR1 cDNA序列；金属亲和层析法纯化原核高效表达的芍药ACS和ETR1蛋白并制备多克隆抗体；qPCR和Western印迹技术分析芍药切花ACS和ETR1基因的时空表达及外源乙烯、乙烯抑制剂STS和1-MCP、伤害处理及糖诱导对ACS和ETR1基因表达的影响，分析芍药ACS和ETR1基因表达与切花衰老的相关性；构建组成型表达启动子CaMV 35S及花特异表达启动子pAtAP3融合反义ACS及ETR1基因植物表达载体并转化烟草，在转录水平和蛋白表达水平上检测烟草ACS和ETR1的表达量；从而为有针对性地对芍药切花品种进行基因修饰，培育拮抗乙烯的芍药切花新种质提供理论依据。

中文关键词： 芍药；切花；乙烯；ACS；ETR1

英文摘要： Cut peony (Paeonia lactiflora Pall.) has great value in ornamental application. However, its industrialization is severely affected by lagged post-harvest preservation technology and mechanism of senescence in cut peony. In this study, full-length cDNAs encoding ACC synthase, key gene in ethylene biosynthesis, and ethylene receptor1 (ETR1) will be isolated by RACE from peony cultivars, whose ethylene metabolism had been characterized previously as analogical climacteric type and analogical cultivar which increase during later stage. Based on metal affinity chromatography, the heterologous ACS and ETR1 protein expressed high-level in Escherichia coli will be purified to be used as antigen to produce the polyclonal antibody. The expression of ACS and ETR1 in cut peony will be quantified during different development stages and in different tissues by qPCR and Western blotting. The effect of exogenous ethylene, ethylene inhibitors STS and 1-MCP, wounding and sugar, on expression of ACS and ETR1 and the correlation of ACS and ETR1 expression with the senescence of cut peony will be analyzed. The plant expression vector fusing the constitutive Cauliflower Mosaic Virus 35S promoter or flower-specific expression promoter pAtAP3 with antisense ACS and ETR1, respectively, will be constructed and be transformed into Nicoti

英文关键词： Paeonia lactiflora；cut flower；ethylene；ACC synthase；Ethylene receptor 1