解析水稻抗铝毒蛋白ART1的调控机理以及通过遗传筛选克隆水稻抗铝毒新基因

Aluminum (Al) toxicity is a major limiting factor for crop production on acidic soils, which comprise approximately 21% and 40% of China's and the world's arable land, respectively. Rice is the most Al resistance species among small grain cereal crops, but our understanding on the mechanisms of its high Al resistance is still limited. Recently, a transcription factor-encoding gene ART1 was identified, which was demonstrated to be involved in Al resistance in rice. ART1 regulates the expression of downstream Al resistance genes to detoxify Al in the presence of Al. Nevertheless, in the absence of Al, ART1 did not regulate the expression of the Al resistance genes. These suggest that other proteins interacting with ART1 are required for the activation of ART1 function in Al resistance in the presence of Al. To identify interaction proteins of ART1, we plan to perform yeast two-hybrid screening.After several rounds of screening and confirmation, we will select some candidate genes for further functional analysis. We will use a combination of mutant analysis and genetic, physiological, and molecular biological approaches to examine the function of the genes. In addition, to identify more genes involved in Al resistance in rice, we are going to screen Al sensitive mutants by using a mutant library of the reference cultivar Nipponbare irridated with γ rays. Based on a prelimiary screening on a portion of the mutant library, we isolated two mutants sensitive to Al. We will use the Al sensitive mutants and map-based cloning approach to clone the corresponding Al resistance genes. Then we will use various approaches to study the function of the genes. Our study will not only contribute to our understanding of high Al resistance mechanisms in rice, but also provide a basis for genetic engineering of Al resistance genes to improve Al tolerance in crops in the future.

酸性土壤占我国21%和世界40%以上的耕地面积,而铝毒是作物在酸性土壤生产的主要限制因子。水稻是小粒谷物中最抗铝毒的作物，但其它高抗铝毒机制仍不十分清楚。近来一个特异参与解除铝毒的转录因子ART1被鉴定出来，它在铝毒存在的情况下通过转录调控下游抗铝毒基因的表达来调节对铝毒的抗性。ART1在无铝毒时并不参与调控抗铝毒基因的转录,这说明在有铝毒时存在其它蛋白与ART1相互作用使其发挥作用。本项目准备通过酵母双杂交技术筛选和ART1相互作用的蛋白，然后结合突变体分析和其它遗传、生理和分子生物学等各种手段对ART1互作蛋白进行详尽的功能解析。除此此外，通过对水稻突变库进行铝敏感突变体的筛选，已分离到两个铝敏感突变体，计划利用这些突变体材料和图位克隆技术克隆抗铝毒基因，然后通过各种技术手段对它们进行功能解析。以此加深对水稻抗铝毒机制的了解。

酸性土壤占我国21%和世界30%以上的耕地面积,而铝毒是作物在酸性土壤生产的主要限制因子。水稻是小粒谷物中最抗铝毒的作物，近年来若干个水稻抗铝毒基因相继被克隆和功能解析，较大地推动了水稻抗铝毒机制的研究，但是仍有许多未知的机制待解析，包括抗铝毒转录因子ART1的调控机制、铝毒在细胞内的信号转导、铝毒受体、铝在细胞内的螯合形态等。本研究通过筛选与ART1的互作蛋白以及筛选铝毒敏感或铝抗突变体，并对克隆的基因进行功能研究来进一步剖析水稻的抗铝毒机制。通过酵母双杂交技术，我们获得10个与ART1互作的蛋白，并对其中的两个同源的互作蛋白OsVOZ1和OsVOZ2作了进一步的功能分析，研究结果表明这两个蛋白是水稻抗铝毒的负调控因子并且功能冗余，可能通过调控ART1基因的表达来影响水稻的抗铝毒。通过筛选约5000个水稻突变株系，我们获得5个铝毒敏感和1个铝抗新颖突变体。我们对铝抗突变体ral1和铝毒敏感突变体sal1、sal2作进一步的研究。我们的研究结果表明，ral1对铝毒的抗性主要由于其根尖细胞壁积累更少的铝造成，而该突变体结合更少的铝可能是由于果胶质含量减少导致。通过基因定位结合全基因组重测序，我们克隆了RAL1基因，该基因编码一个参与木质素合成的酶。sal1突变体对铝、铜、镧都更敏感，并且表现对低温超敏感，这提示SAL1参与多个抗逆过程。SAL1基因被定位在第4染色体的长臂上。sal2突变体对铝、铜、钙都敏感，但对镉和镧不敏感。我们克隆了SAL2基因，它编码一个磷酸酶，这暗示SAL2可能参与细胞内铝毒的信号转导过程。本项目研究结果揭示了水稻新的抗铝毒机制，并为提高作物抗铝毒提供新思路。