黄瓜韧皮部汁液响应盐胁迫的蛋白质组学及抗性相关基因功能分析

Cucumber is a cosmopolitan important vegetable, also the model plant of vascular system research, and sensitive to salt stress. Protein is executant of physiological function and direct embodiment of life phenomenon. Plant phloem is the important constituent of vascular system, which consists of a large number of proteins related with stress. In this study, cucumber is used as experiment material. The phloem sap is used as the research object, which includes mass protein. Two-dimensional gel electrophoresis and mass spectrometry techniques based on proteomics are utilized for protein separation and identification responded to salt stress in cucumber phloem sap protein. Diversity protein and synthesis of special protein are investigated. Combining on bioinformatic approach, characteristics of function protein was analyzed by western blot and real-time fluorescent quantitative PCR techniques. The function protein in the phloem sap is confirmed. Subsequently, genes of the confirmed function protein located in phloem sap are cloned, which are introduced into arabidopsis and transgenic plants were obtained. Phenotype, salt tolerance, growth and physiological characteristics in transgenic plants are studied. The responses of molecular mechanism in phloem sap to salt stress are primarily explained. Therefore, this study possesses important meanings to clarify the molecule response to salt stress in cucumber, adaption mechanism,discovery of specific markers and improvement of stress tolerance, and enrichment of plant stress physiology theory.

黄瓜是世界性的重要蔬菜，也是维管系统研究的模式植物，对盐渍环境敏感。蛋白质是生理功能的执行者和生命现象的直接体现者。韧皮部作为维管系统重要组成部分，内含大量与胁迫相关蛋白。本研究以黄瓜为试材，以富含蛋白质的韧皮部汁液为研究对象，利用以双向电泳和质谱分析为基础的蛋白质组技术，分离和鉴定响应盐逆境的韧皮部汁液蛋白，研究盐胁迫对韧皮部汁液蛋白差异表达、特异蛋白合成影响；结合生物信息学方法并采用Western blot和实时荧光定量PCR技术分析盐胁迫下的差异蛋白，明确响应盐胁迫的韧皮部汁液差异蛋白特性；克隆经分析确定响应盐胁迫的韧皮部汁液差异蛋白基因，采用农杆菌介导法转入拟南芥，研究盐胁迫下转基因植株的表型和抗盐性，初步探明响应盐胁迫韧皮部汁液差异蛋白功能。该研究对解析黄瓜在盐胁迫下的分子响应和适应机理、寻找用于黄瓜耐盐性评价的特异标志物、改良黄瓜的抗盐性以及丰富植物逆境生理理论等均有重要意义。

本研究以黄瓜盐敏感品种‘津优1号’和较耐盐品种‘新泰密刺’为材料，采用营养液栽培，通过正常营养液和75 mmol NaCl胁迫处理，在处理第3天时收集各处理植株的韧皮部渗出液，通过SDS-PAGE电泳检测发现韧皮部液中提取的蛋白纯度较高，所有提取样品适于进行下一步二维液相色谱分级分离实验。通过同位素相对标记与绝对定量蛋白质定量技术（iTRAQ技术）鉴定到745个韧皮部渗出液蛋白，与对照处理相比，盐胁迫显著调节了两个黄瓜品种中111个蛋白的表达，在盐敏感品种中发现有69个蛋白差异表达，在较耐盐品种中发现有65个蛋白差异表达，其中，有23个差异蛋白是两个品种中共调节蛋白。对这些盐逆境响应蛋白质的功能进行分类，结果表明，它们在黄瓜的各种新陈代谢过程中都有出现，大部分检测到的功能类型包括：蛋白质转换、分子伴侣、碳水化合物的运输和代谢、能源生产和转换、翻译、转录、氨基酸的运输和代谢、信号转导机制等。利用实时荧光定量PCR技术，分析9个盐逆境响应的韧皮部渗出液蛋白的mRNA表达水平，验证蛋白表达与mRNA表达的关系。克隆过氧化物酶蛋白基因，并进行序列和时空表达分析，结果显示，该基因的ORF长度为1035 bp，编码344个氨基酸，与其他植物过氧化物酶相比表现出了很高的相似性，时空表达分析表明该基因具有潜在的响应盐胁迫的生态功能。克隆黄瓜韧皮部渗出液差异蛋白钙调素类1蛋白（Calmodulin-like protein 1-like，CML1）基因，并成功构建其表达载体，通过花序侵染法介导转化拟南芥，获得转CML1基因拟南芥植株。转基因拟南芥植株的抗盐能力增强、生长速度加快；与野生型植株相比，盐胁迫下转基因植株种子发芽率较高，植株长势较好，根长受抑制程度显著降低。本研究初步明确了黄瓜韧皮部渗出液响应盐胁迫的蛋白质组变化，研究了所筛选的差异蛋白基因的表达特性和功能，对解析黄瓜适应盐胁迫的分子机制以及丰富植物逆境生理理论等具有重要意义。