重金属胁迫下植物类萌发素蛋白的生理作用

Germin-like proteins (GLPs) which have similar sequence to wheat germins are soluble glycoproteins and ubiquitous in land plants. GLP family members possess superoxide dismutase (SOD), oxalate oxidase (OXO) and polyphenol oxidase (PPO) activities and play other physiological roles. In our previous study, GLP expression in rice are up-regulated at transcriptional and protein levels by heavy metals including copper and cadmium. In the present project, we will focus on the GLP genes responding to heavy metal stresses, and isolate and characterize the GLP genes and the promoter region. The heavy metal-inducible activity of the promoter and localize the heavy metal-regulatory will be investigated by deletions analysis of the promoter. The enzymatic activities and characteristics of the GLP protein with SOD, OXO or PPO will be studied in vivo and vitro. The expression characterization of GLPs in plants and its relationships to reactive oxygen species production, biosynthesis of lignin and accumulation and distribution of heavy metals in different plant tissues will be examined in details using OsGLPs-expressing transgenic plants, rice OsGLPs mutant and the complementary lines. Our objectives are 1) to determine the biological functions of GLPs in plants under conditions of heavy metal stress; 2) to further understand molecular mechanisms of heavy metal resistance in plants; and 3) to supply new approach to genetic improvement of heavy metal tolerance and decreasing heavy metal accumulation in edible parts of plants.

类萌发素蛋白（GLPs）是一类广泛存在于植物体内的可溶性糖蛋白，具有超氧物歧化酶、草酸氧化酶和多酚氧化酶等多种酶活性、受体和结构蛋白等生理功能。我们前期研究结果显示，水稻OsGLP基因转录水平和蛋白质表达水平在镉和铜处理下显著提高，但其生理功能仍不清楚。本项目拟从水稻中筛选出应答重金属胁迫的OsGLPx作为研究的目标蛋白，克隆OsGLPx基因及其上游启动子区序列，通过启动子截断缺失分析该基因的转录表达特性及定位。以目标蛋白的酶学特性为重点，利用体内和体外方法研究重金属胁迫对其活性的影响；应用OsGLPx基因过表达、敲除突变体及突变体回补的转基因材料，分析OsGLPx与水稻组织中活性氧积累、木质素沉积、重金属积累等的关系，揭示OsGLPs在植物抗重金属胁迫中的作用。这对阐明植物重金属抗性机制具有重要理论意义，并为降低重金属在植物可食部分中的积累提供新的技术途径。

类萌发素蛋白（GLPs）是一类广泛存在于植物体内的可溶性糖蛋白，具有超氧物歧化酶、草酸氧化酶和多酚氧化酶等多种酶活性、受体和结构蛋白等生理功能。本项目研究了水稻OsGLP4-1、OsGLP8-7和OsGLP8-11具有SOD的酶学活性，并以多聚体的形式存在。过表达OsGLP4-1、OsGLP8-2、OsGLP8-7和OsGLP8-11基因，可降低由重金属诱导引起的活性氧积累。明确OsGLP8-7基因上游启动子区-600~-1000bp存在顺式作用元件与转录因子OsWRKY72结合，负调控OsGLP8-7表达。进一步利用多种遗传材料，揭示了OsGLP4-1、OsGLP8-2、OsGLP8-7和OsGLP8-11都可通过调控木质素沉积来调节细胞壁对重金属的截留作用，从而影响植物对重金属的耐性及积累。本研究还意外发现8号染色体上OsGLP8-2至OsGLP8-11十个串联基因中的OsGLP8-2，更易受Cu、Cd诱导表达，其上调幅度大于其它串联基因。并利用多种研究手段证实了OsGLP8-7蛋白可分别与过氧化物酶POD、几丁质酶CHIT7相互作用。另外，因OsGLP8-7突变影响水稻GST、PCS1及WAK11基因的表达，故研究了GST、PCS1及WAK11在重金属胁迫下的生物学功能。本项目的研究结果对阐明植物重金属抗性机制具有重要理论意义，并为降低重金属在植物可食部分中的积累提供新的技术途径。