苹果生长素响应因子MdARF5的克隆与功能鉴定

doi:10.3864/j.issn.0578-1752.2018.07.012

摘要/Abstract

摘要： 【目的】分离苹果生长素响应因子MdARF5（Auxin Response Factor 5），分析其对生长素的响应，鉴定其在调节花青苷合成过程中的作用，揭示MdARF5的生物学功能，为进一步研究生长素对花青苷的调节提供理论依据。【方法】以‘嘎拉’苹果（Malus×domestica ‘Royal Gala’）为材料，利用同源克隆技术，克隆得到一个ARF（Auxin Response Factor）转录因子，并将其命名为MdARF5。利用MEGA5.0软件构建多物种间系统进化树。通过农杆菌介导的遗传转化获得转基因苹果愈伤组织。比较野生型和转基因苹果愈伤组织花青苷积累的差异。利用烟草叶片瞬时转化试验，分析MdARF5对MdMYB1的转录调控。【结果】克隆获得苹果生长素响应因子MdARF5（序列号：MDP0000143749），该基因CDS为2 691 bp，编码含有896个氨基酸的蛋白。系统进化树分析表明，苹果MdARF5与梨PbARF5同源性最高。基因表达分析显示，该基因响应生长素处理，并且与花青苷合成相关基因表现出相反的表达模式。在苹果愈伤组织中超表达MdARF5，其花青苷积累较野生型显著降低，表明MdARF5在调控花青苷积累过程中发挥重要作用。对苹果MdMYB1启动子序列进行分析，发现其序列包含一个MdARF5的结合位点。烟草瞬时表达试验显示，MdARF5能够抑制MdMYB1的表达。【结论】推测苹果MdARF5可能通过直接抑制MdMYB1的表达负调节花青苷的积累。

关键词: 苹果, 生长素, ARF转录因子, 花青苷

Abstract: 【Objective】The objective of this study is to isolate an apple auxin response factor gene MdARF5, to analyze its expression of exposing to auxin, to identify its role in regulating anthocyanin biosynthesis, then to reveal its biological functions and to provide a theoretical basis for auxin-mediated anthocyanin accumulation. 【Method】 The apple auxin response factor gene MdARF5 was cloned by PCR technology from apple (Malus×domestica ‘Royal Gala’). The phylogenetic tree was constructed by MEGA 5.0 software. The transgenic apple calli were generated via Agrobacterium-mediated transformation. The differences in the anthocyanin accumulation were compared between wild-type and transgenic apple calli. The transient expression assays in tobacco leaves were carried out to test the transcriptional regulation of MdMYB1 gene by MdARF5. 【Result】MdARF5 gene (MDP0000143749) was obtained. The open reading frame (ORF) of MdARF5 contained 2 691 bp, encoding a protein of 896 amino acid residues. Phylogenetic tree analysis showed that the homology of MdARF5 was close to the PbARF5. The transcriptional analysis results indicated that MdARF5 was induced by auxin treatment. On the contrary, the expression levels of anthocyanin biosynthesis genes were repressed. The MdARF5-overexpressing apple calli exhibited decreased anthocyanin content, suggesting that MdARF5 gene might play an important role in regulating anthocyanin accumulation. The sequence of MdMYB1 promoter region was analyzed and a putative ARF binding motif was found. Meanwhile, the transient expression assays were performed in Nicotiana benthamiana leaves and the results showed that MdARF5 could repress the expression of MdMYB1. 【Conclusion】It is speculated that MdARF5 down-regulates anthocyanin accumulation by directly repressing the transcript of MdMYB1.

Key words: apple, auxin, ARF transcription factor, anthocyanin

安建平，宋来庆，赵玲玲，由春香，王小非，郝玉金. 苹果生长素响应因子MdARF5的克隆与功能鉴定[J]. 中国农业科学, 2018, 51(7): 1345-1352.

AN JianPing, SONG LaiQing, ZHAO LingLing, YOU ChunXiang, WANG XiaoFei, HAO YuJin. Cloning and Functional Characterization of an Auxin Response Factor Gene MdARF5 in Apple[J]. Scientia Agricultura Sinica, 2018, 51(7): 1345-1352.

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