鹰嘴豆甘/脯氨酸富集蛋白（CarGPRP1）调控干旱胁迫反应的分子机制研究

Crop production is frequently hindered by environmental stresses such as drought, high salinity and low temperature. Genetic engineering is a significant option for improving crop yield under drought stress, which relies on well understanding of mechanism of plant resistance to dehydration and availability of useful stress-related genes. We identified a chickpea gene coding for glycine and proline-rich protein (GPRP) from a drought-related cDNA library. GPRP family has been barely investigated although its members probably play important roles during plant development and response to environmental stimuli. Our previous study, for the first time, revealed that: CarGPRP1 containing trans-membrane domain was located on both membrane and nucleus; Expression of CarGPRP1 was regulated by various abiotic stresses; Overexpression of CarGPRP1 didn't hinder plant growth but promoted development of lateral roots and enhanced tolerance to abiotic stresses, including drought, salt and cold, in Arabidopsis. It is noteworthy that CarGPRP1 only up-regulated expression of stress-resistant genes under drought stress. In this project, we will firstly study mechanism and functional significance of subcellular localization of CarGPRP1 through deletion mutants and transgenic Arabidopsis. Further, we will identify interactive proteins of CarGPRP1 involved in regulation of stress-related gene expression and investigate their characterization and physiologic functions by a series of assays including protein interaction experiments, transcriptional analysis and transgenic Arabidopsis. We hope to clarify how GPRP regulates expression of stress-related genes under drought stress. Final purpose for this project is to help people understand functional mechanism of GPRP genes and apply them in drought-resistant genetic engineering for crops.

干旱严重影响作物生产，抗旱遗传工程意义重大。作物耐旱机理的解析程度和优质抗旱基因的数量局限着抗旱遗传工程的发展。项目组先前从耐逆鹰嘴豆的干旱cDNA文库中分离到一个甘/脯氨酸富集蛋白编码基因（CarGPRP1）。其所在家族功能重要但鲜被研究。前期实验首次揭示，含跨膜域的CarGPRP1同时分布于细胞膜和核中，转录受到多种逆境信号调节，异位表达不妨碍拟南芥的正常生长却促进侧根发育并增强植株抗旱、盐、冷的能力。更值得注意的是，CarGPRP1只在干旱胁迫下才增强抗逆基因的表达。本项目拟通过缺失突变体和拟南芥转基因实验阐明CarGPRP1亚细胞定位机理及功能意义，并通过蛋白互作、转录分析、反向遗传学等技术，鉴定抗逆基因表达调节相关的CarGPRP1互作蛋白及其特性和生理功能，以期阐明CarGPRP1在干旱生理中调节抗逆基因表达的机制，为GPRP家族的功能解析和工程应用奠定基础。

干旱严重影响作物生产，耐旱新基因的鉴定及分子机理解析有助于制定田间干旱应对措施及培育作物抗旱种质。我们前期从鹰嘴豆干旱cDNA 文库中分离到一个功能未知的甘/脯氨酸富集蛋白编码基因 (CarGPRP1)， 该基因转录受到多种逆境及相关激素的调节，其异位表达可显著增强植株抗旱、盐、低温的能力。GPRP共有的XYPP重复结构也存在于PGYRP和annexin家族中，这些XYPP超级家族成员的表达均受到逆境信号的调节，因而代表了一类新的抗逆基因家族，极具探索价值。然而，XYPP家族成员的抗旱作用机理尚属未知。本项目以CarGPRP1在拟南芥中的高度同源性基因AtGPRP3为主要对象开展研究，获得了几个突破性发现。作为核蛋白，AtGPRP3广谱性表达在各个组织中，但主要集中在莲座叶及茎生叶上。过量表达AtGPRP3会导致植株生长减缓，而抑制其表达会加速植株的生长，这种变化会在互补材料中得到消除，说明该基因是植株营养生长的负调控者。该基因表达受到干旱诱导及ABA抑制，其过量表达可增强植株干旱存活率，而抑制表达降低干旱存活率。缺失体植株中干旱生理相关基因表达均发生了显著变化，例如CCS、RbohF、RbohD、CAT3、NAC3以及P5CS1等。酵母实验杂交鉴定到了代表28种蛋白的35个AtGPRP3互作蛋白。进一步研究证实CAT2 （catalase）、CAT3、CLC3（lysophospholipases）、CYSD1（sulfate adenylyltransferase）可在体外及体内与AtGPRP3结合，其中CAT2和CAT3位于细胞核和质中，而CLC3和CYSD1位于细胞核。CAT具有降解过氧化氢的能力，因而与逆境生理密切相关。无论是ph5.8还是ph8.0的1/2MS培养基上，CAT2表达被抑制的植株均表现营养生长减缓的趋势。这些结果表明GPRP可负向调节CAT来降低营养生长速率并提高抗旱性。本项目验证了XYPP家族成员的抗旱生理功能，首次构建了XYPP蛋白的互作因子文库，首次揭示了XYPP蛋白与其他因子的互作及生理功能意义，从而阐明了以GPRP为代表的XYPP蛋白在抗旱逆境功能的分子机理。这些成果为人们深入理解植物干旱适应机理提供了全新的知识，并为逆境生理学科开启了一个全新的领域，也为作物抗逆种质改良提供了有效目标。