【作者】 王璐；

【导师】 寿惠霞；

【作者基本信息】 浙江大学 ， 植物学， 2013， 博士

【摘要】 铁是植物生长所必需的微量元素。土壤中的铁含量丰富,但铁在土壤中的低溶解性使得它能被植物吸收利用的部分极少,同时过量的铁素也会造成植物铁中毒。研究植物的缺铁响应分子机制,对植物适应缺铁和铁过量等环境逆境,培育优良富铁品种等具有重要的意义。本研究在水稻中克隆了一个碱性螺旋-环-螺旋(bHLH)家族的转录因子——OsbHLH133. OsbHLH133定位在细胞核中,在根中受缺铁强烈诱导,通过对启动子接GUS报告基因的转基因株系的GUS活性分析,发现缺铁时OsbHLH133主要在整个根部细胞,特别是中柱以及地上部分的维管组织中表达。系统进化树表明,在进化上,OsbHLH133与拟南芥、水稻和番茄中已被报道的参与缺铁响应分子机制的bHLH转录因子位于不同的分支中,表明OsbHLH133极有可能具有不同于已知的铁相关的bHLH蛋白的新功能。通过对OsbHLH133超表达转基因植株(OsbHLH133-OE)和T-DNA插入突变体(bhlh133)的研究,发现缺铁时bhlhl33突变体叶片叶绿素含量(SPAD)上升,而OsbHLH133-OE植株的SPAD值下降。与野生型植株相比,突变体植株的地上部分铁含量升高,而根中铁含量降低。OsbHLH133-OE植株的铁含量测定结果与之相反,即地上部分铁含量降低,而根中铁含量上升。此外,OsbHLH133-OE植株木质部液中的铁含量也低于野生型。由此可见,OsbHLH133参与调控铁素在水稻地上部分和根中的分布。基因芯片结果结合定量RT-PCR分析表明,缺铁响应基因在根和叶中的表达与铁含量相关联,即铁含量高的组织,缺铁响应基因的表达低,铁含量低的组织,基因的表达被诱导,缺铁响应基因的表达直接反应了植株的局部铁含量水平。另外,芯片分析结果显示有很多信号通路相关的基因在突变体和野生型根中的表达具有显著差异,这些基因包括钙离子信号通路中的相关基因,说明了钙离子信号在植物的铁平衡过程中可能发挥作用。本研究旨在完善植物缺铁响应信号途径的理论基础,缺铁诱导乙烯合成也是植物响应缺铁的一种表现,本论文初步开展了缺铁诱导乙烯合成的分子机理研究。通过对拟南芥中的9个有ACC合成酶功能的ACS基因的表达分析,发现AtACS2/6/7/11不论在叶中还是在根中都受到缺铁显著诱导,这表明了拟南芥中这几个ACS基因很可能参与缺铁诱导乙烯合成这个过程中。此外,AtACS2/6的上游调控蛋白AtMPK3/6的转录表达也受缺铁诱导。不同缺铁时间的乙烯含量分析表明,缺铁诱导拟南芥的乙烯合成,而诱导倍数最高为1.7-2倍。由于拟南芥ACS家族基因功能冗余严重,我们无法在供缺铁乙烯含量仅2倍的差异条件下,通过ACS基因相关突变体的乙烯含量测定来筛选负责缺铁诱导乙烯合成的ACS基因。通过对拟南芥mpk3和mpk6突变体乙烯含量的测定,发现缺铁时,mpk6植株的乙烯合成量显著低于野生型,而mpk3的乙烯合成量也有所降低,但不显著,这表明AtMPK6很可能参与调控缺铁诱导乙烯合成这个过程。综上所述,本论文对水稻OsbHLH133转录因子进行了研究,发现OsbHLH133的表达受缺铁强烈诱导,功能研究表明OsbHLH133调控着铁在水稻地上部分和根部的分布,很有可能参与调控铁在木质部中的长距离运输。通过对拟南芥AtACS基因,AtMPK基因相关突变体的研究,本论文初步确定了参与缺铁诱导乙烯合成的ACS基因,而AtMPK6可能参与调控这个过程。更多还原

【Abstract】 Iron is an essential mineral element for plant growth. Although iron (Fe) is abundant in the earth’s crust, it is not available to plants because the low solubility of Fe in soil solution. On the other hand, excess accumulation of Fe can cause damage to plant cells. Studying on the molecular mechanism in response to Fe deficiency is very important for plants to adapting to the stress caused by Fe deficiency or Fe excess accumulation. It also plays a significant role in developing crop varieties with improved iron content.In this study, we characterized a novel basic helix-loop-helix (bHLH) transcription factor in rice, named OsbHLH133. OsbHLH133was targeted to the nucleus, and the transcript abundance of OsbHLH133was strongly up-regulated in roots under Fe-deficiency conditions. Using transgeneic rice expressing the GUS reporter gene driven by the OsbHLH133endogenous promoter, it was found that OsbHLH133is expressed throughout the whole roots, preferently in the stele of the roots and vascular budndles of the shoots in response to Fe deficiency. Phylogenetic analysis showed that OsbHLH133is not closely related to other known Fe-responsive bHLH transcription factors. Thus, OsbHLH133should be involved in an unknown mechanism related to Fe homeostasis.Study on OsbHLH133overexpression (OsbHLH133-OE) lines and T-DNA insertional mutant(bhlh133) plants showed that the leaf chlorophyll content (SPAD value) increased in bhlh133mutant while decreased in OsbHLH133-OE plants. Compared to wild type (WT), bhlh133showed growth retardation with enhanced Fe concentration seen in shoots and reduced Fe concentration in roots. OsbHLH133-OE had the opposite effect, which is resulted in an enhanced Fe concentration in roots and reduced Fe concentration in shoots. In addition, Fe concentration in xylem sap of OsbHLH133-OE also reduced. Therefore, alteration of the transcript abundance of OsbHLH133affected Fe distribution between roots and shoots.Microarray and quantitative RT-PCR analysis showed that the transcript abundances of some of the genes encoding Fe-related functions in OE, bhlh133and WT plants were related to the local Fe concentration. Expression of Fe deficiency response genes was repressed in the tissue with higher Fe concentration, and was Induced in the tissue with lower Fe concentration. Significant differential expression of a number of signalling pathways, including calcium signalling, was also seen in bhlh133plants compared to WT. It suggested a role for calcium signalling in Fe homeostasis of plants.The main purpose of this study is to improve the regulatory mechanism of Fe deficiency response in plants. In response to Fe-deficiency, ethylene production was induced. The preliminary study on the molecular mechanism of ethylene synthesis induced by Fe deficiency was conducted in this study. Analysis of the transcirpt abundances of the9authentic ACS genes in Arabidopsis showed that AtACS2/6/7/11was up-regulated by Fe deficiency in the leaves and roots. It suggested those four ACS genes might be involved in the pathway of ethylene synthesis which was induced by Fe deficiency. Additionally, the expression of AtMPK3and AtMPK6which can phosphorylate AtACS2/6was also induced by Fe deficiency.Time-course analysis of ethylene production under different period of Fe deficiency indicated that ethylene production was induced by Fe deficiency in Arabidopsis, and the induction level was up to1.7-2folds. Because of the function reductant of ACSs and less than2folds of ethylene production induced by Fe deficiency, it is difficult to identify the ACS which is involved in biosynthesis of ethylene that induced by Fe deficiency by measuring the ethylene production of ACS mutants. Analysis of the ethylene production of mpk3and mpk6mutant plants showed a significantly reduced ethylene production in mpk6and a slight reduced in mpk3compared to WT under Fe-deficiency condition. It suggested a role for AtMPK6in regulating ethylene production which is induced by Fe deficiency.Taken together, we characterized a novel bHLH transcription factor, named OsbHLH133in rice. This transcription factor is strongly up-regulated by Fe deficiency. Our results indicated that OsbHLH133acted as an important regulator of Fe distribution between shoots and roots and it might be involved in regulating the root-to-shoot Fe translocation. The ACS genes involved in the ethylene biosynthesis which is induced by Fe deficiency were identified in Arabidopsis and AtMPK6might be the regulator of this pathway.更多还原