腾冲红花油茶花蜜抑菌蛋白筛选及其功能验证

花蜜蛋白生态功能是当今花蜜化学研究中最为活跃的领域。近年来，研究者相继从一些植物花蜜中分离出各种蛋白，并证明其中一些具有抑菌功能。研究表明模式植物烟草的花蜜蛋白通过氧化还原循环机制能抑制花蜜中微生物生长。然而已证实这一机制并非存在于所有植物中。我们前期研究表明，腾冲红花油茶花蜜总蛋白含量较高，且粗提蛋白能显著抑制油茶腐败花蜜中微生物生长，但目前有关该花蜜抑菌蛋白组成及其抑菌机制尚不清楚。因此，本项目拟以腾冲红花油茶花蜜为研究对象，1）利用双向电泳和质谱结果并结合生物信息学分析，明确微生物侵染下花蜜中差异表达蛋白的种类、注释其功能并筛选出候选抑菌蛋白；2）针对候选抑菌蛋白，利用酶活实验确定花蜜抑菌蛋白，利用克隆技术构建原核表达体系表达纯化抑菌蛋白并验证其抑菌效果；3）通过qRT-PCR技术检测花蜜抑菌蛋白基因响应微生物侵染的动态表达特征，以揭示腾冲红花油茶蜜腺组织防御微生物侵染的作用机制。

The ecological functions of nectar proteins have become the most active fields among the recent nectar biochemistry studies. Understanding the biochemical mechanisms by which plants respond to microbial infestation on nectar is a fundamental goal of plant pollinator biology. Previous studies have demonstrated that a variety of floral nectar proteins have been isolated from some plant nectar, and some of them have defensive functions against microbial invasion. The nectarins in the nectar of ornamental tobacco have been biochemically characterized and are likely to protect nectar from microbial infestation through the Nectar Redox Cycle. However, the novel biochemical mechanism is not found in all plants floral nectar. Our previous results had demonstrated that abundant proteins secreted into the nectar of Camellia reticulata had the function of anti-microorganism. Both these protein components and the mechanism of defense microbial infestation are still unclear. Therefore, this project is aimed at these aspects: (1) identify the nectar proteome against microbial infestation through the 2-DE, MALDI-TOF MS/MS, analyze functions of differently expressed proteins in the floral nectar and select candidate antimicrobial proteins by bioinformatics analysis; (2) explicate the antimicrobial functions of candidate antimicrobial protein by detecting enzyme activity in floral nectar and SDS PAGE gel, obtain antimicrobial protein by constructing the prokaryotic or eukaryotic expression system through the technology of clone, then confirm the effects of antimicrobial protein on microorganism growth; (3) use Real-time Quantitative PCR to determining the temporal expression of genes of antimicrobial proteins in the floral nectary when the floral nectar is under microbial infestation. This study will provide data to further understand the ecological roles of nectar proteins and fully elucidate the possible biochemical mechanism of nectar proteins defending microbial infestation in camellia reticulata.