番茄slebf2基因的克隆及遗传转化研究-cloning and genetic transformation of tomato sle bf2 gene.docxVIP

摘要乙烯是一种重要的植物激素，对植物生长发育及果实成熟过程具有重要的调 控作用。在拟南芥中研究表明，EBF2 基因编码的 F-box 蛋白，通过泛素/蛋白酶体 途径降解乙烯信号转导通路中的重要调控因子 EIN3 蛋白参与调节植株对乙烯的反 应。但因拟南芥的果实发育与成熟特征不明显，故目前在拟南芥中获得的结果并 不能阐明 EBF2 蛋白对番茄等肉质果实发育与成熟的影响。为了揭示 EBF2 在果实发育及成熟过程中的功能，本研究克隆并鉴定了番茄Sl-EBF2 基因，进行了番茄遗传转化研究。主要研究结果如下：（1）采用 RT-PCR 方法扩增出 Sl-EBF2 基因。测序结果显示，Sl-EBF2 基因 全长编码区为 1914 bp，编码 637 个氨基酸序列。亚细胞定位研究显示其定位于细 胞核内。（2）采用定量 PCR 方法对 Sl-EBF2 基因的表达模式分析表明，Sl-EBF2 基因 在花中、开花期及果实发育的破色期和后熟期均有较高表达水平。（3）构建了植物超表达载体 pCambia1301-EBF2，经 PCR 和测序验证正确后 转化入农杆菌 GV3101。（4）采用农杆菌介导的叶盘法转化番茄，在 Kan 100 mg/L 的筛选压力下获得 抗性植株。（5）采用 PCR 和 GUS 染色方法对所获得的番茄抗性植株进行转基因鉴定， 共获得 3 株阳性植株，表明目的基因已整合到番茄基因组中。进一步通过半定量 RT-PCR 分析证实，Sl-EBF2 基因在转基因番茄植株中被超量表达。（6）与野生型对照植株的表型比较分析发现，转基因植株表现出生长缓慢，叶片颜色变深，部分果实长期处于黄果状态，用乙烯处理黄果不转红，且果实多 呈无籽或少籽等现象。关键词：番茄，Sl-EBF2 基因，克隆，遗传转化ABSTRCTEthylene is an important plant hormone in regulating plant development and fruit ripening. It has been proved that in Arabidopsis, F-box proteins play an important role in ethylene signaling parthway through directing EIN3 for degradation by the Ubiquitin/proteasome, while EIN3 is a member of regulators in ethylene pathway. However, results in Arabidopsis could not elucidate the effect of EBF2 protein on development and ripening in fleshy fruit such as tomato (Solanum lycopersicum), since Arabidopsis don’t have significant features of maturing.In order to clarify the function of EBF2 in fruit development and ripening,Sl-EBF2 genes were cloned from tomato, and then transformed in tomato. The main results are as follows:Sl-EBF2 gene was amplified by RT-PCR. Sequencing result reveals that the fulllength of the code region is 1914 bp, encoding 637 amino acids. Subcellular localization exhibits nuclear targeting of Sl-EBF2 protein.Expression patterns analysis by Quantitative-PCR shows that there arehigh-level expressions in flower, during anthesis, break and ripening of fruit.Over-expression vector pCambia1301-EBF2 was constructed and verified by PCR and sequencing then transformed into Agrobacterium GV310