【作者】 郑录庆；

【导师】 寿惠霞；

【作者基本信息】 浙江大学 ， 植物学， 2009， 博士

【摘要】 铁是维持植物正常生命活动的重要微量元素。虽然土壤中富含铁素,但是由于铁的可利用性非常低,因此植物缺铁现象相当普遍。同时,由于植物是人类与其他动物铁营养的主要来源,研究并阐明植物中铁的吸收、转运、代谢、储存等机制,对于提高全球粮食产量和改善人类营养意义重大。磷是影响植物铁的吸收利用及其有效性的一个重要因素,铁磷元素之间存在的生理拮抗互作是植物营养研究领域备受关注的热点。为探明磷对于水稻铁的吸收利用的影响及铁磷互作的分子机制,本研究对苗期水稻植株进行不同铁磷营养的供给处理（+Fe+P,-Fe+P,+Fe-P,-Fe-P）,分析了在不同磷铁供给水平下水稻幼苗的生理表现和基因表达的变化。研究发现,虽然水稻植株中的总铁含量在两种缺铁处理中（-Fe+P,-Fe-P）都显著的降低,但是水稻植株只在-Fe+P处理条件下表现为显著缺铁症状。进一步对四种处理条件下的植株水溶性铁含量的分析结果显示相对于-Fe+P处理条件,-Fe-P条件下水稻植株中有效铁含量显著提高,表明缺磷能够显著提高植株中铁的有效性。这一结论在两种+Fe条件下同样适用,即+Fe+P条件下铁的有效性显著的低于+Fe-P条件。以上结果更明确了植物铁磷拮抗互作的普遍性。利用Affymetrix水稻全基因组芯片对上述四种不同铁磷水平处理的水稻根及地上部分的基因表达的转录本分析得到如下结果。首先,基因表达的主成分分析与基因差异表达数都表明,相对于单种元素的缺乏（缺铁或缺磷）,铁磷双缺与正常培养条件的基因表达谱相似度较高,说明铁磷间存在的拮抗互作关系。其次,与+Fe+P条件相比,-Fe+P条件下产生了总共7,628个显著性（P＜0.05）差异表达基因,而这些基因中仅有约15%在-Fe-P条件下仍存在显著性差异。说明缺磷能够很大程度上回复缺铁响应基因的差异性诱导表达。此外,通过上述基因差异表达分析,发现了许多参与铁的吸收与转运,缺铁响应基因的表达调控基因以及代谢途径相关基因等缺铁响应基因,其中不仅包含了一些已知的缺铁响应基因（例如IRO2,OsIRT1,OsNAS1,OsYSL15等）,还发掘了许多未有报道的新的缺铁响应基因,例如OPT转运体家族基因OsOPT1与OsOPT2,bHLH家族转录因子OsIRbHLH1与OsIRbHLH2等。对这些新发现的缺铁响应基因功能的研究将大大提高我们对植物缺铁响应过程的了解,也将为这些基因在分子育种中的应用提供理论基础。在上述新发现的缺铁相应基因中,我们选取一个在水稻根及地上部分都显著受缺铁上调表达的bHLH转录因子OsIRbHLH2进行了功能的初步研究。通过各种缺素处理下基因表达分析表明该基因在水稻根及地上部分受缺铁特异响应,该基因与sGFP的融合表达亚细胞定位结果表明OsIRbHLH2蛋白定位于细胞核内,可能为转录因子。对OsIRbHLH2超表达转基因水稻的缺铁响应表型及基因表达的分析表明,OsIRbHLH2对水稻缺铁响应基因的表达有负调控效应,过量表达OsIRbHLH2造成转基因水稻在低铁条件下不能有效的诱导缺铁响应机制,从而表现出对缺铁超敏感的现象。对植物缺铁响应机制及铁的动态平衡的研究的一个重要目标是应用于培育富铁作物来解决目前全球性缺铁流行的问题,本研究利用基因工程技术与铁生物有效性评价的细胞模型筛选并发现了一种新的能够促进铁吸收的成分物质-尼克酰胺（Nicotianamine,NA）。水稻种子中特异超表达NA合成酶基因OsNAS1后获得的NA高积累的转基因水稻,在人结肠癌细胞系（human colonadenocarcinoma cell line,Caco-2）细胞模型评价体系中,其籽粒的铁吸收效价显著高于原始品种。用化学合成的NA添加到米粉中,同样能够显著地提高米粉中铁的有效性,NA与抗坏血酸（AA）添加至FeSO₄与FeCl₃中的对比实验表明,NA对铁有效性的促进作用高于AA。综上所述,我们发现了一种新型的植物来源的铁吸收促进剂NA,为生物强化解决全球性缺铁问题提供了一种切实有效的策略。更多还原

【Abstract】 Fe is an essential micronutrient for plants.Although Fe is one of the most abundant elements in the Earth’s crust,Fe deficiency is a worldwide agricultural problem on calcareous soils with low-Fe availability.On the other hand,because plant based food is the main diet for peoples from many developping areas,Fe deficiency is the most prevalent micronutrient deficiency.Therefore,understanding of the machanisms of the iron uptake,transport,metabolism,storage and their regulation is not only important for the improvement of crop production,but also for the development of crop varieties with densed Fe to alleviate the world-wide Fe deficiency problem.Interaction between Fe and P has been noted in the field of plant nutrition.To understand the physiology and molecular mechanisms of their interaction,we studied the growth performance,nutrient concentration,and gene expression profiles of root and shoot segments derived from 10 day old rice seedlings under four different nutrient conditions;ⅰ) full strength of Fe and P,(+Fe+P),ⅱ) full strength of P and no Fe(-Fe+P),ⅲ) full strength of Fe and no P(+Fe-P) andⅳ) without both Fe and P (-Fe-P).While removal of Fe in the growth medium resulted in very low shoot and root Fe concentrations,the chlorotic symptoms and retarded seedling growth were only observed on seedlings grown in the presence of P.Microarray data showed that in roots,7,628 transcripts were significantly changing in abundance in the absence of Fe alone.Interestingly,many of these were changes were reversed if P was also absent (-Fe-P),with only～15%overlapping with-Fe alone(-Fe+P).Analysis of the soluble Fe concentration in rice seedling shoots showed that P deficiency resulted in significantly increased Fe availability within the plants.The soluble Fe concentration under -Fe-P conditions was similar to that under +Fe+P conditions.These results provide evidence that the presence of P can affect Fe availability and in turn can influence the regulation of Fe responsive genes. The microarray analysis identified many Fe deficiency response genes,including many classical -Fe response genes,such as IRO2,OsIRT1,OsNAS1,OsYSL15,and many novel genes with unknown functions.These genes includes OPT transport family genes OsOPT1 and OsOPT2,bHLH transcription factors OsIRbHLH1 and OsIRbHLH2 and so on.Among these novel Fe regulated genes,the bHLH-domain contained putative transcripofion factor OsIRbHLH2 was studied.Nutrient deplete treatments show that the expression of OsIRbHLH2 was specifically induced by Fe deficiency conditions,but not by other nutrients(-N,-P,-K,-Zn,-Mn,-Cu). OsIRbHLH2 protein is localized into nucleus,supporting that OsIRbHLH2 is an putative transcription factor.The physiological and molecular responses of the OsIRbHLH2-overexpressing transgenic rice lines suggest that OsIRbHLH2 is a negative regulator in Fe deficiency responses in rice.Development of crop varieties with improved iron content and bioavailiablility is important approach for the alleviation of Fe deficiency problem world-wide.In this study,rice nicotianamine(NA) synthase gene(OsNAS1) was endosperm-specifically expressed in rice grain.The resulted transgenic rice accumulated a significant amount of NA in the grains.Bioavailability of iron from the high NA grain,as measured by ferritin synthesis in an in vitro Caco-2 cell model that simulates the human digestive system,was twice as much as that of the control line.When added at 1:1 molar ratio to ferrous iron in the in vitro system,NA produced two times more ferritin than that of ascorbic acid(AA),one of the most potent enhancers of iron bioavailability.These data demonstrated that NA is a novel and powerful promoter of iron utilization. Biofortifying polished rice with this compound has great potential in combating global human iron deficiency in people dependent on rice for their sustenance.更多还原