硒缓解黄瓜镉毒害生理机理及特异蛋白和相关基因的研究

Soil cadmium (Cd) contamination poses a severe detrimental influence on environment, and plant, animal and human health. In this study a series of experiments will be conducted with the main objectives as follows: To elucidate physiochemical mechanism of alleviation effects of selenium application on cadmium toxicity in cucumber, by characterizing physiochemical characters, including anatomic structure of roots, ATPase and antioxidant enzyme activities, and Cd uptake and subcellular distribution using hydrponic and pot experiments of exposing cucumber to Cd, Se, Cd and Se levels; To identify stress-specific proteins for Cd toxicity tolerance in cucumber using two-dimensional gel electrophoresis and mass spectrometry and bio-informatics, based on the analysis of the effect of Cd toxicity on the expression of soluble proteins through modifying and optimizing the key procedures, such as sample preparation/dissolution, protein extraction, IEF electrophoresis, SDS -PAGE and dyeing routine; Meanwhile, with the aid of degenerate primers design based on the N-terminal partial sequence from identified Cd-stress-specific protein to amplify the corresponding cDNA by rapid amplification of cDNA ends (RACE), followed by databases homologous searches, a full length cDNA of Cd tolerance-relevant gene will be hopefully sequenced. These results will be valuable for illustrating the molecular mechanism in Cd tolerance and providing theoretical basis and technology support for the agricultural application of exogenous Se alleviates cucumber Cd toxicity and for the cucumber safe production.

土壤镉污染对环境、动植物及人体健康有极大的危害。本研究以栽培黄瓜津研四号为材料，硒缓解黄瓜水培镉胁迫试验，通过形态解剖与生理生化研究，探讨质膜及液泡膜ATPase、抗氧化酶活性与耐镉性和镉积累等的关系，阐明硒对黄瓜镉吸收、分配与转运的影响及缓解镉毒的生理与生化机制；利用IEF/SDS-PAGE双向电泳、质谱肽质量指纹谱分析和生物信息学等技术，比较硒缓解镉毒害后蛋白质组表达的改变，分离鉴定耐镉及低镉积累特异蛋白质；依据目标特异蛋白N端部分氨基酸序列设计简并引物，RACE扩增，快速、简便获得包含完整编码区的全长cDNA，结合应用生物信息学等技术进行功能预测，转基因功能验证，筛选克隆耐镉及低镉积累相关基因。研究结果将完善黄瓜耐镉毒机制的研究理论，并为利用基因工程技术构建耐镉低镉积累植物奠定分子生物学基础和技术平台，为硒缓解黄瓜镉毒害的农业应用和黄瓜安全生产提供理论依据和技术支撑。

黄瓜是一种世界性的重要蔬菜，也是我国北方地区种植范围最广、面积最大的蔬菜种类之一；土壤镉污染对环境、动植物及人体健康具有极大的危害。本项目以栽培黄瓜津研四号为材料，黄瓜土培盆栽及水溶液培养实验，模拟硒、镉、镉+硒胁迫处理，通过根系和叶片的解剖结构、光合作用、活性氧代谢、碳水化合物代谢、ATPase、抗氧化酶活性及重金属吸收与分布等性状分析，探讨硒对黄瓜镉吸收、分配与转运的影响及缓解镉毒害的生理与生化机制；结果表明外源硒通过改善黄瓜细胞结构、光合作用，减少黄瓜叶片和根系镉诱导产生的ROS含量，降低膜脂过氧化产物MDA，缓解镉胁迫下黄瓜中产生的氧化胁迫，同时缓解镉胁迫引起的碳水化合物含量及元素含量的不平衡，从而降低黄瓜镉含量、缓解镉毒害。利用IEF/SDS-PAGE双向电泳、质谱肽质量指纹谱分析和生物信息学等技术，比较硒缓解镉毒害后蛋白质组表达的改变，分离鉴定耐镉及低镉积累特异蛋白质，它们涉及胁迫、代谢、光合作用和贮藏相关蛋白，包括谷胱甘肽S-转移酶F8，热休克蛋白STI，过氧化物酶，醛缩酶-二磷酸果糖2，铁氧还蛋白-亚硝酸还原酶和抗坏血酸氧化酶。利用气相色谱-质谱联用（GC-MS）技术，明确了黄瓜代谢组对镉毒害的响应及外源硒缓解镉毒害响应的特异代谢物，叶片中有丙酮酸、乳糖、乳酸、草酸等，根系中主要有α-D-氨基葡萄糖-1-磷酸、葡萄糖-6-磷酸1、葡萄糖-6-磷酸2、6-磷酸葡萄糖酸、木糖醇、5-磷酸核酮糖1等；同时，结合应用生物信息学等技术进行功能预测，筛选耐镉及低镉积累相关基因，研究结果可丰富黄瓜耐镉的机理理论，为缓解黄瓜镉毒害的农业应用和黄瓜安全生产提供理论依据和技术指导。