水稻金属耐受蛋白OsMTP8转运Mn2+的分子机制研究

Manganese (Mn) is an essential micronutrient for plants. It is of particular importance in photosynthesis as the catalytic center for light-induced water oxidation in photosystem II, and is required as a cofactor for a variety of enzymes, such as decarboxylases and numerous glycosyl transferases, and also is a constituent of mitochondrial Mn2+-dependent superoxide dismutase (MnSOD) . In plants, Mn toxicity is a widespread phenomenon on acid soils because soil Mn becomes more available at low pH and in reducing conditions. On acid soils, Mn toxicity together with Al toxicity, is a main limiting factor for agriculture production. Recently, some of the genes responsible for Mn transport in plants have been identified at the molecular level. These include transporters responsible for Mn accumulation into the cell and release from various organelles, and for active sequestration into endomembrane compartments, particularly the vacuole and the endoplasmic reticulum. Several transporter gene families have been implicated in Mn transport, including cation/H+ antiporters, natural resistance-associated macrophage protein (Nramp) transporters, zinc-regulated transporter/iron-regulated transporter (ZRT/IRT1)- related protein (ZIP) transporters, the cation diffusion facilitator (CDF) transporter family, and P-type ATPases. In the rice plant, there are several transporter genes have been found in Mn transport, these genes include OsNRAMP5, OsYSL6, OsYSL2 and some of other genes. The metal tolerance proteins (MTP) belong to the cation diffusion facilitator (CDF) transporter family, they are important for maintenance of cation homeostasis in bacteria, yeast, plants, and mammals. In general, MTPs play a critical role in removing transition metals from the cytosol, either by intracellular sequestration or by export from the cell. There are 10 members of MTPs in rice plant. So far, there is only one member of MTPs in rice was investigated, which is OsMTP1. It is localized in the cell membrane, and is necessary for efficient translocation of Zn, Cd and other heavy metals, and maintain ion homeostasis in plant. The other MTPs in rice remains to be investigated. Our recent results showed that expression of the OsMTP8 in a manganese hypersensitive yeast mutant restores manganese tolerance to wild-type levels. Subcellular localization of OsMTP8 in onion epidermal cells showed that it localized to the vacuole membrane. These results suggested that OsMTP8 is a vacuole membrane localized transpoter which responses for the Mn transport in rice plant. Building on our recent results, we will determine (a) cation specificity in rice plant; (b) intracellular and cellular localisation in planta; (c) function in planta with mutants and overexpressors to investigate the OsMTP8 function. The identification of the gene responsible for Mn transport will substantially improve our understanding of plant Mn homeostasis.

锰是重要的植物微量营养元素，但酸性土壤中锰的活性提高，易对植物产生毒性。金属耐受蛋白MTP (Metal Tolerance Protein)主要负责植物体内金属离子的转运，能将金属离子转运到胞外或者区室化到亚细胞结构内以达到缓解其毒害的目的。我们前期通过酵母突变体功能互补实验发现水稻金属耐受蛋白OsMTP8在酵母中具有转运锰离子而不是其他离子包括铜、镉、锌、钴的功能，洋葱表皮细胞定位显示其表达在液泡膜上，提示OsMTP8可能通过将锰离子储存在液泡内而提高水稻的耐锰性。因此本项目将通过基因表达分析，蛋白亚细胞定位，组织水平表达分析，突变体、RNAi和转基因超表达水稻植株的生理水平分析等实验手段对OsMTP8的功能进行深入的研究，明确该基因在水稻应答锰胁迫、吸收和转运等过程中的功能，为阐明植物细胞中锰离子吸收与转运的调控机制提供理论依据，并进一步扩展植物微量元素研究的内涵。

锰是重要的植物微量营养元素。在酸性土壤中二价锰的浓度会增高，从而对植物正常生长产生毒性。金属耐受蛋白 MTP (Metal Tolerance Protein)主要负责植物体内金属离子的转运，能将金属离子转运到胞外或者区室化到亚细胞结构内以达到缓解其毒害的目的。通过酵母突变株互补试验说明OsMTP11具有转运锰离子的功能，结合过表达、干扰OsMTP11的植株以及T-DNA插入的突变体的表型分析和离子含量测定，进一步验证了OsMTP11具有转运锰离子的功能。在水稻原生质体以及烟草表皮细胞中的表达以及相关抑制剂处理，发现OsMTP11定位于反面高尔基体网状结构上。在锰胁迫的情况下，OsMTP11能从反面高尔基体网状结构迁移到质膜上去，推测OsMTP11通过胞吐的方式把锰离子转运到细胞膜外。以上结果显示OsMTP11是水稻中的一个锰离子转运蛋白，能够通过外排的方式减少水稻中锰离子的含量来达到减缓水稻受到锰毒害的水平。同时过表达OsMTP11相当于野生型水稻更能耐受锰毒害，为水稻中锰毒害的研究提供了理论基础以及为今后的水稻育种提供了思路和材料储备。通过对OsMTP8.1的关键氨基酸残基进行定点突变和随机突变，然后利用酵母功能互补的方法对OsMTP8.1转运离子的活性进行研究，发现了一些关键的氨基酸残基参与到OsMTP8.1转运离子活性的发挥，并通过蛋白质三维结构模拟出了这些关键氨基酸残基的位置以及可能参与离子转运活性发挥的机制。. 已发表SCI 收录论文 3 篇，其中共同通讯作者一篇，共同第一作者一篇，均标注受本基金项目资助，两篇影响因子均大于 5，另有一篇SCI论文投稿中。