【作者】 陈丽茜；

【导师】 郑绍建；

【作者基本信息】 浙江大学 ， 植物学， 2012， 硕士

【摘要】 铁是植物生长发育必需的微量元素。由于土壤中铁的生物有效性普遍较低,缺铁是限制全球农业生产的重要因素之一。植物缺铁时会产生各种生理和分子机制来增加土壤中铁的生物有效性和增强根部的铁吸收能力。番茄缺铁时,先通过高铁还原酶FRO1将Fe3+还原成Fe2+,后Fe2+经转运蛋白IRT1转运入根部。FER编码bHLH蛋白,是一类转录因子,在转录水平和转录后水平调控FRO1和IRT1。目前对于FER是如何感知缺铁信号、如何受到调控以及高铁还原酶活性(FCR,Ferric Chelate Reductase)如何受到调控知之甚少。TFT7基因的表达受到缺铁诱导,为了进一步了解其分子调控机理,我们采用病毒诱导的基因沉默(VIGS)的方法研究了14-3-3基因(TFT7)在番茄缺铁响应中的作用。用烟草花叶病毒介导的14-3-3基因(TFT7)沉默载体,以农杆菌为媒介侵染番茄获得基因沉默植株。结果表明,在缺铁条件下,14-3-3基因(TFT7)沉默后,根部高铁还原酶活性明显降低,同时FRO1基因表达也下调,侧根根尖膨大和根毛的形成受到抑制。该结果证明了14-3-3蛋白在转录水平和转录后水平调控缺铁诱导的高铁还原酶活性,并且能促进根尖膨大和根毛的形成。14-3-3基因(TFT7)沉默后,FER基因的表达量也相应下调。有趣的是,番茄T3238fer突变体中,无论是正常或缺铁处理下,TFT7基因表达量都下调。通过酵母双杂技术,发现二者并不能互作。因此,我们推测TFT7、FER之间存在反馈调节机制来互相调节表达。另外,我们认为可能有第三种因子能与TFT7、FER或者分别与其中之一相互形成复合物来调控下游的响应。综上,我们证明了14-3-3基因(TFT7)确实在缺铁响应中起作用,在转录水平和转录后水平调控缺铁诱导的高铁还原酶活性,促进根尖膨大和根毛的形成,与FER互相反馈调控或者与FER和(或)第三种因子形成复合物调控缺铁信号途径。更多还原

【Abstract】 Iron is one of the essential micro-nutrients for plant, and iron (Fe) deficiency constitutes one of the major limitations for crop production in calcareous soils. Plants employ a number of responses to Fe deficiency including some specific physiological and molecular actions that can either increase Fe bioavailability in the soils or enhance the root capacity to uptake Fe. In tomato, reduction of ferric iron is mediated by iron reductase FRO1 and subsequent uptake of ferrous iron into root epidermis by the metal transporter IRT1. The bHLH transcription factor FER is required for the regulation of FRO1 and IRT1. However, the signaling pathway leading to the activation of ferric chelate reductase (FCR) activity or FER action still remains unclear. TFT7 was induced following the imposition of Fe starvation. We utilized VIGS (virus-induced gene silencing) to individually reduced the expression of TFT7, which resulted in the reduce of FCR activity and the expression FRO1 in response to Fe deficiency, and the suppression of the root tip swelling and root hair formation, suggesting that TFT7 regulates FCR activity on transcription and post-transcription level.In TFT7 silenced lines, FER expression was suppressed either in normal or iron defficiency condition. Interestingly, in fer mutant line, TFT7 expression was also repressed and its transcriptional response to Fe deficiency was eliminated. Yeast two-hybrid assay indicated that TFT7 could not interact with FER. It was speculated that there is a positive feedback loop between TFT7 and FER to which they mutually regulate each other’s expression. Or maybe there is another protein to binding both,then to trigger iron deficiency response.Taking all of these findings together, we demonstrated that TFT7 takes part in regulating the signaling pathway of Fe-deficiency-induced FCR activity, promoting the root tip swelling and root hair formation and has a positive feedback loop with FER.更多还原