柑橘枳雀砧木耐缺铁的生理与分子机制研究

It is known that selecting proper rootstocks is a good method for improving nutrient use efficiency. Zhique, a citrus rootstock originated in China, shows strong acclimation to limestone matrix-developed alkaline soil. Our preliminary research revealed that the acclimation of Zhique is due to the tolerance to iron deficiency, while the relevant mechanisms remain unclear. In this project, the Zhique and Trifoliate orange (A common citrus rootstock susceptible to iron deficiency) were used as a pair of comparable materials to study the iron deficiency tolerant mechanisms in citrus. We will first investigate the root architecture and anatomical structure and available Fe content in these two rootstocks upon iron deficiency treatment, by using our established hydroponic or sand-pot culture system. Meanwhile, the mycorrhizal fungi community structure and dominant / iron-related fungus will be identified in pot experiment using alkaline soils from producing area. Then, we will examine the sequences and spatiotemporal patterns of transcription expression of genes involved in iron activation(HA), Fe3+ reduction (FRO) and Fe2+ absorption (IRT/NRAMP) upon iron deficiency using quantitative RT-PCR methods. Further analysis of enzymatic or transport activities by yeast mutant complement experiments may lead to identification of key genes in relation to iron absorption. Finally, we will attempt to investigate the biological function of these key genes by over-expressing the iron-related genes from Zhique using Trifoliate orange transformation system. Results from this study will not only help understand the physiological and molecular mechanisms underlying iron deficiency tolerance in Zhique rootstock, but also facilitate the research and application towards enhancing iron utilization in citrus.

通过选择砧木来提高果树养分利用效率是公认的有效途径。枳雀是原产我国的珍贵柑橘砧木资源，对石灰岩为母质的碱性土壤有较强的适应性，前期研究也表明其具有很强的耐缺铁能力，但其机制尚不清楚。本项目拟以枳雀和枳为材料，采用水培/砂培盆栽来研究其在缺铁胁迫下根系构型和解剖结构的差异，并测定盆栽植株各部位的有效铁含量差异，以弄清其基本的生理机制；同时又通过土培盆栽（缺铁产区土壤）来研究砧木间在菌根菌的群落组成差异，筛选优势菌根菌并研究其耐缺铁的作用。在此基础上，采用定量PCR等方法研究在铁活化、Fe3+还原、Fe2+吸收过程中的HA、FRO、IRT/NRAMP等基因在枳雀和枳中的序列和表达差异，以此来筛选其关键基因成员，采用酵母突变体互补实验验证铁运输载体的活性，进一步通过枳遗传转化体系解析关键基因在铁吸收中的功能。由此探明枳雀砧木耐缺铁的生理与分子机制，为抗性资源的挖掘和提高铁利用效率提供依据。

枳雀是原产我国的珍贵柑橘砧木资源，在碱性土壤上表现出极强的耐缺铁能力，其机制尚不清楚。本项目以耐缺铁性差异极大的枳雀和枳为材料，采用水培/砂培盆栽研究了其生理和分子水平的差异及机制。主要结果表明：（1）缺铁条件下枳雀根系的氢离子分泌量和氢离子流速显著高于枳，枳雀砧有更强的吸收土壤铁的能力。（2）在铁的活化、Fe3+还原和Fe2+转运（铁吸收过程）环节中，负责氢离子分泌的关键基因HA6在枳雀中的表达显著高于枳，功能验证表明HA6基因显著促进枳砧对铁的吸收；同时发现缺铁诱导的转录因子MYB308仅在枳雀中结合HA6的启动子并激活其表达，从而使得枳雀具有了耐缺铁的机制。（3）在铁吸收的3个环节中，Fe3+还原和Fe2+转运过程，枳雀根系的高铁还原酶活性和FRO2的表达差异不显著或还较低；枳雀IRT1的表达量较低但对缺铁的响应较快。（4）铁转运关键基因IRT1启动子有MITE1/2插入，研究表明该MITE的存在与柑橘耐缺铁的遗传背景有密切关系，由此开发分子标记用于鉴定耐缺铁性的柑橘资源。（5）菌根的差异研究表明，枳雀的耐缺铁性并不依赖于菌根和土壤微生物，而枳砧在一定程度上需要土壤微生物改善，由此建立了柑橘根际微生物资源库并挖掘到一株假单胞菌能显著促进柑橘耐缺铁。