苹果MdWRKY18和MdWRKY40参与盐胁迫途径分子机理研究

doi:10.3864/j.issn.0578-1752.2018.23.010

摘要/Abstract

摘要：

【目的】研究苹果WRKY转录因子MdWRKY18和MdWRKY40蛋白结构、表达水平及在盐胁迫中的功能,为进一步完善盐胁迫分子机理的研究提供参考。【方法】以‘红脆2号’苹果为试材,克隆MdWRKY18和MdWRKY40,对其蛋白结构进行分析;采用qRT-PCR测定该基因在盐胁迫条件下的表达水平,并通过GUS染色分析它们启动子活性,利用酵母双杂分析其互作关系,并通过转基因验证其功能。【结果】蛋白结构分析表明MdWRKY18和MdWRKY40均含有一个WRKY、Cx5C以及HxH结构域;MdWRKY18和MdWRKY40表达水平和启动子活性受150 mmol·L ^-1 NaCl诱导,酵母双杂交试验表明,MdWRKY18和MdWRKY40能够和自身互作形成同源二聚体,且MdWRKY18也能和MdWRKY40互作形成异源二聚体;在‘王林’愈伤中分别过表达MdWRKY18和MdWRKY40时,能够促进MdSOS1和MdNHX1的表达,并提高‘王林’愈伤在盐胁迫处理下的生长量,在‘王林’愈伤中共表达MdWRKY18和MdWRKY40时,同样能够促进MdSOS1和MdNHX1的表达,但对提高‘王林’愈伤的生长量要高于分别过表达MdWRKY18和MdWRKY40。【结论】苹果MdWRKY18和MdWRKY40受盐胁迫的诱导,可以形成同源或异源二聚体,并增强‘王林’愈伤对盐胁迫的耐性。

关键词: 苹果, WRKY转录因子, 盐胁迫, GUS染色, 酵母双杂交

Abstract:

【Objective】 In order to improve the molecular mechanism of salt stress, we studied several aspects of MdWRKY18 and MdWRKY40 in apple WRKY transcription factors, including the protein structure, the expression level and the function in salt stress. 【Method】 We cloned the MdWRKY18 and MdWRKY40 genes in ‘Hongcui No.2’ apple and analysed their protein structure. The expression levels of MdWRKY18 and MdWRKY40 were studied by the qRT-PCR under the salt stress, and their promoter activities were analyzed using the GUS staining. We analyzed the interaction relationship between MdWRKY18 and MdWRKY40 proteins by yeast two-hybrid and verified their function by transgenosis. 【Result】 Analysis of protein structure showed that both MdWRKY18 and MdWRKY40 proteins contained a WRKY, Cx5C and HxH structural domains. The expression levels and promoter activities of MdWRKY18 and MdWRKY40 were induced by the 150 mmol·L ^-1 NaCl. The yeast two-hybrid experiments showed that MdWRKY18 and MdWRKY40 could respectively interact with itself to form homodimers, and MdWRKY18 could also interact with MdWRKY40 to form heterodimers. When MdWRKY18 and MdWRKY40 was overexpressed respectively in orin callus, they could increase the callus weight under salt stress and promote the expression of MdSOS1 and MdNHX1. When MdWRKY18 and MdWRKY40 were co-overexpressed in orin callus, it could also promote the expression of MdSOS1 and MdNHX1, however, the weight of callus was heavier than the weight of callus overexpressing MdWRKY18 or MdWRKY40. 【Conclusion】 MdWRKY18 and MdWRKY40 were induced by the salt stress, and they could form homodimers or heterodimers, overexpressing MdWRKY18 or MdWRKY40 in orin callus could increase its salt tolerance.

Key words: apple, WRKY transcription factor, salt stress, GUS staining, yeast two-hybrid

许海峰,杨官显,张静,邹琦,王意程,曲常志,姜生辉,王楠,陈学森. 苹果MdWRKY18和MdWRKY40参与盐胁迫途径分子机理研究[J]. 中国农业科学, 2018, 51(23): 4514-4521.

XU HaiFeng,YANG GuanXian,ZHANG Jing,ZOU Qi,WANG YiCheng,QU ChangZhi,JIANG ShengHui,WANG Nan,CHEN XueSen. Molecular Mechanism of Apple MdWRKY18 and MdWRKY40 Participating in Salt Stress[J]. Scientia Agricultura Sinica, 2018, 51(23): 4514-4521.

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