中文摘要：

【目的】克隆苹果中的两个ERF（ethylene responsive factor）转录因子，对其序列与表达进行分析；并进一步构建原核表达载体，在大肠杆菌中诱导融合蛋白表达，为解析两基因的抗病功能及作用机制奠定基础。【方法】首先，将抑制性消减杂交筛选到的EST序列进行BLAST比对，根据比对获得的MdAP2D4与MdAP2D19的cDNA序列设计引物进行克隆；然后，利用MEGA4．1软件将两个ERF蛋白与拟南芥的AP2家族蛋白进行聚类分析，对保守的hP2功能域的氨基酸序列进行分析；并进一步利用RT-PCR检测两基因对外源MeJA的响应；最后，将两个基因分别连接到原核表达载体PCEX-4T-1上，利用IPTG对转化的大肠杆菌BL21进行诱导。【结果】MdAP2D4与MdAP2D19两个基因均与拟南芥B3组ERF亲缘关系最近，在叶片中表达较高，都能被外源的MeJA诱导表达。SDS-PAGE检测结果表明，两个基因的融合蛋白均能够被IPTG诱导表达。【结论】MdAP2D4与MdAF2D19为B3组ERF转录因子，外源MeJh能够诱导其表达。

英文摘要：

[Objective] Cloning, sequence and expression analysis of two ERF （ethylene responsive factor） transcription factors from Malus domestica Borkh and expression of their fusion proteins in E. coli were determined to identify their disease resistant function and molecular mechanism. [Method] ESTs derived from suppression subtractive hybridization were blasted, and two EFR genes, MdAP2D4 and MdAP2D19, were cloned which contain these ESTs; Subsequently, phylogenetic relationship including the two apple proteins and ,4rabidopsis AP2 domain-containing proteins was analyzed using MEGA4.1 and the multiple alignment of AP2/ERF domain was constructed; Additionaly, the in vitro shoot cultures of ＇Gala＇ apple were used to identify the two genes expression in response to exogenous MeJA; Finally, the two genes were inserted into vectorpGEX-4T-1, and IPTG was used to induce the fusion proteins expression in E. coli BL2I. [Result] The phylogenetic relationship of MdAP2D4 and Md,4P2D19 was closer with Arabidopsis ERF proteins of group B3 than others. The transcript levels of MdAP2D4 and MdAP2D19 were higher in leaves than other organs and could be induced by exogenous MeJA. The results of SDS-PAGE demonstrated that the fusion proteins of the two genes could express in E.coli BL21 induced by IPTG. [Conclusion] MdAP2D4 and MdAP2D19 are ERF transcriptionfactors belonging to group B3, and they can be induced by exogenous MeJA.