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    水曲柳FmPIF基因家族克隆及表达模式分析

    Cloning and expression pattern analysis of FmPIF gene family in Fraxinus mandshurica

    • 摘要:
        目的  探究水曲柳光敏色素互作因子(FmPIFs)在激素调控与非生物胁迫应答过程中的重要作用,为揭示水曲柳抗逆分子机制和制定林木遗传育种策略提供理论依据。
        方法  在水曲柳中克隆FmPIFs基因,并对其基因结构、蛋白质理化性质、保守基序、系统进化关系等进行生物信息学分析。采用qRT-PCR方法分析水曲柳中FmPIFs基因在不同组织及不同激素与胁迫条件下的表达模式。
        结果  获得5个水曲柳FmPIFs基因家族成员,分别命名为FmPIF1、FmPIF3、FmPIF4、FmPIF7和FmPIF8,其对应的蛋白均为亲水性不稳定蛋白,全部定位在细胞核内。多序列比对结果表明FmPIFs均存在APB保守结构域,成员FmPIF1与FmPIF3存在特有的APA结构域。组织特异性分析显示FmPIFs均在叶中表达量最高,其中成员FmPIF8表达量最高,为对照的3.96倍;但在茎中少量表达,茎中表达量最高的是FmPIF3,仅是对照的0.21倍;而在根中表达量均极低。胁迫响应分析表明,FmPIFs正调控水曲柳植株盐、碱和干旱胁迫抗性,而对植株抗寒性起负调控作用,其中成员FmPIF3对寒冷及盐胁迫明显响应,FmPIF8在碱胁迫下表达量明显上调,FmPIF1在干旱胁迫下表达量明显上调。在激素响应结果中,FmPIFs对脱落酸(ABA)、水杨酸(SA)和赤霉素(GA3)的响应较为一致,而对生长素(IAA)和茉莉酸甲酯(MeJA)的响应存在差异。FmPIF1在施加MeJA后剧烈应答且表达量显著上调,FmPIF7在SA处理后表达量明显上调,FmPIF3和FmPIF4在GA3处理后表达量明显上调。
        结论  FmPIFs各成员在基因及蛋白结构上表现出较高的一致性。RT-qPCR结果表明,FmPIFs在水曲柳叶片中表达量最高。在盐、碱、干旱和寒冷胁迫下,FmPIFs被诱导表达,且大部分表达模式相似。FmPIFs也在水曲柳响应IAA、ABA、MeJA、SA、GA3激素调控过程中发挥重要作用。

       

      Abstract:
        Objective  This paper aims to explore the important role of Fraxinus mandshurica phytochrome interaction factors (PIFs) in the process of hormone regulation and abiotic stress response, and provide theoretical basis for revealing the molecular mechanism of Fraxinus mandshurica resistance and formulating forest genetic breeding strategies.
        Method  The FmPIFs gene was cloned from Fraxinus mandshurica, and its gene structure, protein physicochemical properties, conserved motifs, and phylogenetic relationships were analyzed by bioinformatics. The qRT-PCR method was used to analyze the expression patterns of FmPIFs genes in Fraxinus mandshurica in different tissues and under different hormones and stress conditions.
        Result  Five members of FmPIFs gene family of Fraxinus mandshurica were obtained and named as FmPIF1, FmPIF3, FmPIF4, FmPIF7 and FmPIF8. The corresponding proteins were all hydrophilic and unstable proteins, all of which were located in the nucleus. The results of multiple sequence alignment showed that FmPIFs all had APB conserved domains, and members FmPIF1 and FmPIF3 had unique APA domains. Tissue-specific analysis showed that FmPIFs were all expressed in leaves at the highest level, and member FmPIF8 expressed at the highest level, which was 3.96 times of control. However, it was expressed in a small amount in the stem, and the highest expression in stem was FmPIF3, which was only 0.21 time of control. The expression in root was extremely low. Stress response analysis showed that FmPIFs positively regulated the resistance of Fraxinus mandshurica plants to salt, alkali and drought stress, while negatively regulated plant cold resistance. The member FmPIF3 responded significantly to cold and salt stress, and the expression of FmPIF8 was significantly up-regulated under alkali stress, the expression of FmPIF1 was significantly up-regulated under drought stress. In the hormone response results, FmPIFs hadrelatively consistent responses to abscisic acid (ABA), salicylic acid (SA) and gibberellin (GA3), while responses to auxin (IAA) and methyl jasmonate (MeJA) existed difference. FmPIF1 responded violently after MeJA application and its expression was significantly up-regulated, FmPIF7 was significantly up-regulated after SA treatment, and FmPIF3 and FmPIF4 were significantly up-regulated after GA3 treatment.
        Conclusion  FmPIFs show high consistency in gene and protein structure. RT-qPCR results show that FmPIFs express the highest amount in the leaves of Fraxinus mandshurica. FmPIFs are induced to express by salt, alkali, drought and cold stress, and most of the expression patterns are similar. FmPIFs also play an important role in the regulation of Fraxinus mandshurica in response to IAA, ABA, MeJA, SA and GA3 hormones.

       

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