芍药切花ACS和ETR1基因的克隆、时空表达及功能分析

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 Nicotiana tabacum. The expression of ACS and ETR1 in transgenic tobacco, both in transcription and protein expression level, will be analyzed. This project will provide theoretical basis for targeted gene modification on peony cultivar so as to cultivate new cut peony germplasm which can antagonism ethylene.

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。

芍药（Paeonia lactiflora Pall）是中国的传统名花，全世界约有50多个国家在开发芍药的切花生产。‘桃花飞雪’芍药切花整枝切花的乙烯代谢呈末期上升的特征，表明乙烯在芍药切花的开放和衰老进程中具关键调控作用。项目组主要围绕芍药切花乙烯代谢和信号转导关键基因ACS和ETR1开展了基因克隆、表达分析及功能鉴定工作。首先，以‘桃花飞雪’芍药切花露色期、绽口期、初开期、半开期、盛开期、始衰期共6个时期的花瓣混合样品为试材，开展了芍药切花转录组测序研究，组装共获得105655个重叠群，其中含64627个独立基因；共编码39616个蛋白质；共鉴定出5355个潜在的SSR标记和61367个SNP标记；转录组数据已提交至DDBJ序列数据库（DRX027794）。其次，应用RACE和RT-PCR技术，分离了37个编码芍药乙烯代谢关键酶（ACS和ACO）、乙烯信号转导关键元件（乙烯受体家族成员ETR1、ETR2、ERS1、EIN4及EIN3、EIL1、EIL2、EBF1、EBF2、ERF1、ERF2等）或候选内标基因（泛素基因家族成员、肌动蛋白、18s rRNA等）的cDNA序列或DNA序列并已提交至NCBI核酸序列数据库；geNORM和NORMfinder软件综合分析结果表明在14个常用的内标基因中，Actin（JX310002）基因表达最稳定，是芍药切花乙烯代谢及信号转导元件基因进行qPCR分析的适宜内标基因；qPCR分析结果表明芍药ACS和ETR1基因的表达均具组织特异性，ACS和ETR1基因在花托中的表达量均最高，暗示花托是芍药乙烯释放的主要部位之一；ACS基因在始衰期表达量最大，其转录活性与ACS酶活性保持一致；ETR1基因在露色期、盛开期和始衰期花瓣中的表达较高，表明乙烯受体ETR1与芍药切花的开放与衰老均具有重要关联；成功地在大肠杆菌中异源高效表达了芍药Actin、ACO和ACS重组蛋白并获得了具有生物活性的重组芍药ACO蛋白。第三，构建了芍药ACS、ETR1正义及反义植物表达载体，农杆菌介导法转化了烟草，分子鉴定获得了转基因烟草植株，转基因烟草的形态、生理及ACS和ETR1基因的定量表达有待于进一步分析。本项目在分子水平上证实了ACS和ETR1在芍药切花开放衰老进程中起重要作用，是芍药切花衰老的重要调控因子，是芍药切花品种特性分子改良的主要候选基因。