超表达苹果细胞分裂素响应基因MdCRF6影响花青苷积累和盐胁迫抗性

doi:10.3864/j.issn.0578-1752.2017.16.014

摘要/Abstract

摘要： 【目的】克隆苹果细胞分裂素响应因子基因MdCRF6（cytokinin response factor 6），鉴定其在调节花青苷积累和盐胁迫抗性中的作用，揭示MdCRF6的功能，为研究细胞分裂素信号途径和果树生长发育调控提供理论依据。【方法】以‘嘎啦’苹果（Malus domestica‘Gala’）为研究材料，利用同源序列比对和PCR技术，分离细胞分裂素响应基因MdCRF6。使用MEGA 5.0软件构建苹果MdCRF6与拟南芥CRFs间系统进化树；通过SMART软件和DNAMAN软件分析MdCRF6蛋白的保守结构域。利用实时荧光定量PCR方法检测该基因对细胞分裂素和盐胁迫的响应。通过电泳迁移率试验（EMSA），验证MdCRF6原核表达蛋白对DRE作用元件的绑定。构建MdCRF6植物超表达载体，并通过农杆菌介导的遗传转化获得转MdCRF6苹果愈伤组织。比较野生型和转基因苹果愈伤组织在花青苷积累和盐抗性方面的差异，结合基因表达分析，初步鉴定MdCRF6在调节花青苷积累和盐胁迫方面的生物学功能。【结果】分离得到了苹果细胞分裂素响应因子基因MdCRF6（基因序列号：MDP0000783818），该基因开放阅读框（ORF）为1 047 bp，编码含有348个氨基酸的蛋白。进化树分析和氨基酸序列比对结果表明，苹果MdCRF6蛋白在N端包含保守的CRF结构域，在C端包含保守的AP2/ERF结构域，并且与拟南芥AtCRF6同源性最高。基因表达分析显示该基因具有细胞分裂素和盐胁迫响应，分别在10 μmol·L^-1 BA和100 mmol·L^-1 NaCl处理3 h和6 h时表达量最高。EMSA试验结果验证MdCRF6原核表达蛋白能够绑定DRE序列。在苹果愈伤组织中超表达MdCRF6，发现MdCRF6转基因苹果愈伤组织花青苷积累受到抑制，同时苹果愈伤组织的抗盐性降低。基因表达分析显示，MdCRF6显著抑制花青苷合成基因和盐响应相关基因的表达。【结论】苹果MdCRF6与拟南芥AtCRF6具有高度的同源性，其参与植物对细胞分裂素和盐胁迫的响应。在苹果愈伤组织中超量表达MdCRF6抑制其花青苷积累，降低植物抗盐性。推测苹果MdCRF6可能通过结合花青苷合成基因以及抗盐相关基因的启动子，抑制基因的表达，从而负调节花青苷积累和抗盐性。

关键词: 苹果, MdCRF6, 花青苷, 盐胁迫

Abstract: 【Objective】The objective of this study is to clone apple cytokinin response factor gene MdCRF6, to analyze its homology with Arabidopsis AtCRF6, its expression of exposing to cytokinin and salt stress as well as its binding to DRE motif (ACCGAC), to identify its role in regulating anthocyanins accumulation and salt stress, and to provide a theoretical basis for studying cytokinin signaling pathway and regulating fruit tree growth and development. 【Method】 MdCRF6 gene was cloned by homology sequence alignment and PCR technique. The phylogenetic tree of MdCRF6 and Arabidopsis CRFs was constructed using MEGA5.0. The conserved domains of MdCRF6 were analyzed using SMART software and DNAMAN software. Real-time fluorescent quantitative PCR (qRT-PCR) was performed to detect the gene expression of MdCRF6 exposing to cytokinin and salt stress. The electrophoretic mobility shift assay (EMSA) was conducted to verify whether MdCRF6 could bind to the DRE motif. A plant over-expression vector of MdCRF6 was constructed and used to transform apple calli by Agrobacterium-mediated method. The phenotypes of wild-type and transgenic apple calli on anthocyanins accumulation and salt stress tolerance were characterized to investigate the function of MdCRF6 in regulating anthocyanins accumulation and salt stress tolerance in apple. 【Result】 A cytokinin response factor gene named MdCRF6 (MDP0000783818) was cloned from Malus domestic ‘Gala’. Sequence analysis showed that its open reading frame (ORF) was 1 047 bp, which encoded 348 amino acids. The results of phylogenetic tree and amino acid sequence alignment indicated that MdCRF6 contained a CRF domain in its N-terminal side and an AP2/ERF domain in its C-terminal side. qRT-PCR analysis indicated that MdCRF6 was responsive to cytokinin and salt stress, and the expression levels peaked at 3 h and 6 h exposing to 10 μmol?L-1 BA and 100 mmol?L-1 NaCl, respectively. EMSA assay showed that MdCRF6 could bind to the DRE motif. The MdCRF6-overexpressing apple calli exhibited reduced anthocyanins content and decreased salt stress tolerance. The gene expression analysis showed that overexpression of MdCRF6 significantly repressed the expression levels of anthocyanins biosynthetic genes and salt response related genes. 【Conclusion】The apple MdCRF6 exhibited high similarity to AtCRF6, and it was involved in the response to cytokinin and salt stress. Overexpression of MdCRF6 in apple calli inhibited anthocyanins accumulation and decreased salt stress tolerance. It is speculated that MdCRF6 down-regulated anthocyanins accumulation and salt stress tolerance by repressing the expression of anthocyanins biosynthetic genes and salt response related genes.

Key words: apple, MdCRF6, anthocyanins accumulation, salt stres

安建平，宋来庆，赵玲玲，由春香，王小非，郝玉金. 超表达苹果细胞分裂素响应基因MdCRF6影响花青苷积累和盐胁迫抗性[J]. 中国农业科学, 2017, 50(16): 3196-3204.

AN JianPing, SONG LaiQing, ZHAO LingLing, YOU ChunXiang, WANG XiaoFei, HAO YuJin. Effects of Overexpression of Apple Cytokinin Response Factor Gene MdCRF6 on Anthocyanins Accumulation and Salt Stress Tolerance[J]. Scientia Agricultura Sinica, 2017, 50(16): 3196-3204.

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