两个苹果乙烯反应因子MdAP2D4与MdAP2D19的序列分析及原核表达

doi:10.3864/j.issn.0578-1752.2013.17.012

摘要/Abstract

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AP2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PGEX-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MdAP2D19为 B3组ERF转录因子，外源MeJA能够诱导其表达。

关键词: 抑制性消减杂交 , 茉莉酸甲酯 , 乙烯反应因子 , 原核表达 , 抗病性

Abstract: 【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 Arabidopsis 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 vector pGEX-4T-1, and IPTG was used to induce the fusion proteins expression in E. coli BL21.【Result】The phylogenetic relationship of MdAP2D4 and MdAP2D19 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 transcription factors belonging to group B3, and they can be induced by exogenous MeJA.

Key words: suppression subtractive hybridization , Methyl jasmonate , ethylene responsive factor , prokaryotic expression , disease resistance

田义12, 陈可钦1, 安秀红1, 郝红梅2, 刘志3, 丛佩华2, 郝玉金1. 两个苹果乙烯反应因子MdAP2D4与MdAP2D19的序列分析及原核表达[J]. 中国农业科学, 2013, 46(17): 3635-3642.

TIAN Yi-12, CHEN Ke-Qin-1, AN Xiu-Hong-1, HAO Hong-Mei-2, LIU Zhi-3, CONG Pei-Hua-2, HAO Yu-Jin-1. Sequence Analysis and Prokaryotic Expression of Two Ethylene Responsive Factors MdAP2D4 and MdAP2D19[J]. Scientia Agricultura Sinica, 2013, 46(17): 3635-3642.

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