截形苜蓿中结瘤因子水解酶及其多功能同源蛋白的功能分析

Biological nitrogen-fixation of rhizobia in root nodules of legumes is an environmentally friendly alternative for the use of synthetic nitrogen fertilizer. Hence, it is very important to understand molecular mechanisms that allow legumes to distinguish between mutualistic symbionts and plant pathogens. Bacterial infection depends on rhizobial nodulation signals (Nod factors; lipo-chitooligosaccharides), whose levels are controlled by host enzymes. The applicant’s laboratory has identified MtNFH1, a symbiosis-related Nod factor hydrolase of the model legume Medicago truncatula (Tian et al., 2013, Plant Physiology 163: 1179-1190). The enzyme specifically cleaves Nod factors of Sinorhizobium meliloti and is structurally related to defense-related class 5 chitinases, namely MtCHIT5b. The Nod factor hydrolase exclusively releases lipo-disaccharides from Nod factors, but lacks activity towards chitin or non-modified chitin oligosaccharides. A homology model of the Nod factor-enzyme interaction suggests a substrate binding pocket with a distinct fatty acid binding cleft (formed by loops A and B of MtNFH1). In contrast, the chitinase MtCHIT5b does not cleave Nod factors but substitutions of amino acid residues in either loop A or B converts this enzyme into a Nod factor cleaving enzyme (Zhang et al., 2016 submitted). These results support the view that MtNFH1 evolved from MtCHIT5b by gene duplication and subsequent neo-functionalisation. In this proposal, we propose molecular work on the Nod factor hydrolase and related proteins in M. truncatula. Gene knockout plants of M. truncatula will be characterized to analyze the biological role of MtNFH1 and heterofunctional homologs during symbiosis, plant development and plant defense, respectively. In parallel, temporal and spatial expression analysis as well as subcellular localization studies will be performed. We also will biochemically characterize the MtNFH1 homologs with respect to their carbohydrate-protein interactions. Furthermore, we will engineer novel protein versions with modified activities. Finally, we plan to crystallize the Nod factor hydrolase of M. truncatula together with its bound Nod factor substrate in order to confirm the structure predictions obtained by homology modeling. The findings of this project will increase our knowledge on evolution of proteins presumably involved in nodulation, plant development and plant defense.

生物固氮中了解宿主植物识别共生的机制非常关键。根瘤菌的入侵依赖于它的结瘤信号/因子，宿主分泌的酶调控这信号的浓度但其机制仍不清楚。前期我们发表文章报道鉴定出一个结瘤因子水解酶MtNFH1能特异地剪切结瘤因子但却不能水解几丁质，并在结构上与防御相关的V类几丁质酶即MtCHIT5b相似，模型展示它有一个底物结合口袋及一个脂肪酸结合狭缝。而MtCHIT5b不能剪切结瘤因子，但当狭缝处的氨基酸替换后则可。故我们提出MtNFH1是由MtCHIT5b通过基因复制后获得新功能进化而来。本项目拟构建基因敲除植物来探讨该酶及其相关蛋白在共生、植物发育及防御中的功能；研究时空表达和亚细胞定位；分析有关糖和蛋白的互作方式并构造一个新的有活性的蛋白；最后通过蛋白结晶以验证同源建模的预测。所得的结果不仅提高我们对结瘤、植物发育和和防御相关领域的认知，有助于我们更好地理解蛋白进化，还可为扩大宿主范围提供新的理论基础。

豆科植物与根瘤菌互利共生关系的建立取决于一种重要的信号分子―根瘤菌分泌的结瘤因子。结瘤因子是一种脂质几丁质寡聚糖。然而结瘤因子在共生过程的调控机制及其生物学功能一直是亟待解决的问题。在本课题研究中，我们发现了截形苜蓿中能够特异性水解结瘤因子的水解酶MtNFH1，并对其在共生过程中的生物学功能进行了详尽的解析。本课题通过构建基因敲除植物来探讨该酶及其相关蛋白在共生、植物发育及防御中的功能；研究时空表达和亚细胞定位；分析有关糖和蛋白的互作方式并构造一个新的有活性的蛋白；最后通过蛋白结晶以验证同源建模的预测。所得的结果不仅提高我们对结瘤、植物发育和和防御相关领域的认知，有助于我们更好地理解蛋白进化，还可为扩大宿主范围提供新的理论基础。