导入红海榄总DNA的番茄耐盐相关基因的分离及功能验证

Tomato is one of the main vegetable in our country or even in the world, which belongs to the moderate salt sensitive fresh and processed vegetables crops. The market demand is very large，it plays an very important economic value in the agricultural production. Due to the harm of soil salinization becomes more and more serious, which makes the production of culture tomato largely reduce. In our group, the method of pollen tube path has been used to introduce the total DNA of Rhizophora stylosa into tomato; we have obtained the salt tolerance tomato which can be directly irrigated with sea water containing 2.5-3.0% salts after more than 10 years of salt tolerance screening. In this project, we will use this salt-resistant tomato as the research material, and irrigated by sea water directly and fresh water respectively as the test group, at the same time; we desigh a control group, and then we extract and purify the proteins of leaf and root respectively, after that we analyze the protein by two-dimensions electrophoresis, and we can obtain the differential expression proteins which identified by mass spectrometry and with protein database retrieval. Further more, we can ascertain the homologous protein which related to salt-resistance based on the function prediction. The next step, we design degenerate primers based on the homologous sequences, and then we shall clone the full length of cDNA by RACE, construct the plant expression vector andthen transformed into Arabidopsis by mediating agrobacterium tumefaciens.Finally verify the salt tolerance function of transformed plants. This study can broadly build the genome resource bank of salt tolerance plant and also can provide a novel approach for the research and application of salt tolerance transgenic crops, and our study is also very important for us to exploit and utilize the saline-alkali soil and coastal beach.

番茄是我国乃至世界的主要蔬菜之一，属中度盐敏感型鲜食和加工型蔬菜作物，市场需求量大，在农业生产中有重要的经济价值；由于土壤盐渍化的危害日趋严重，使得栽培番茄大量减产。我们利用花粉管通道技术将耐盐植物红海榄总DNA导入番茄，经十几年的耐盐筛选，已培育出能用含盐2.5%-3.0%的海水直接灌溉的耐盐番茄；本项目将采用这种耐盐番茄做材料，用海水直接灌溉和淡水栽培两种方式进行处理，同时设置对照番茄，分别提取纯化根、叶蛋白，进行双向电泳分析，对获得的两种组合的耐盐番茄差异蛋白进行质谱鉴定和用蛋白质数据库检索，根据功能预测确定与耐盐性相关的同源蛋白；根据同源序列设计简并引物，利用RACE技术克隆耐盐相关基因全长cDNA，通过构建植物表达载体、用农杆菌介导转化拟南芥进行耐盐功能验证。本研究对于拓建植物耐盐基因资源库，为耐盐转基因作物的研究应用开辟新的途径，为开发利用盐碱地和沿海滩涂，都具有重要的意义。

番茄是我国乃至世界的主要蔬菜之一，属中度盐敏感型鲜食和加工型蔬菜作物，市场需求量大，在农业生产中有重要的经济价值；由于土壤盐渍化的危害日趋严重，使得栽培番茄大量减产。我们利用花粉管通道技术将耐盐植物红海榄总DNA导入番茄，经十几年的耐盐筛选，已培育出能用含盐2.5%-3.0%的海水直接灌溉的耐盐番茄；本项目采用我们培育的耐盐番茄做材料，用海水直接灌溉和淡水栽培两种方式进行处理，同时设置对照番茄，分别提取纯化根、叶蛋白，进行双向电泳，获得重复性很好的双向电泳图谱，凝胶经ImageMaster 2D platinum软件分析，叶有约800个蛋白点、根有约550个蛋白点，通过软件定量分析对比，叶共有123 个高丰度的差异蛋白点、根共有139 个高丰度的差异蛋白点，对获得的这些耐盐番茄差异蛋白进行质谱鉴定和用蛋白质数据库检索，根据功能预测确定与耐盐性相关的同源蛋白共有32个，与液泡H+-ATPase有关的6个，与氧化物岐化酶有关的9个，与脱氢抗坏血酸还原酶有关的2个，与蛋白激酶有关的10个，与谷胱甘肽过氧化物酶有关的1个，与钙调蛋白结合蛋白有关的1个，与谷胱甘肽-S-转移酶有关的3个，根据同源序列设计简并引物8对；同时提取高质量番茄叶片RNA, 进行反转录成cDNA, 以cDNA为模板，利用新设计合成的引物进行PCR扩增，对扩增产物液泡H+-ATPase基因720bp、2500bp片段，脱氢抗坏血酸还原酶基因1000bp片段进行基因测序，液泡H+-ATPase基因720bp片段测序结果显示百分之九十八基因是相同的，这说明我们克隆的基因是耐盐相关基因。植物表达载体正在构建当中、有待于用农杆菌介导转化拟南芥进行耐盐功能验证。本研究对于拓建植物耐盐基因资源库，为耐盐转基因作物的研究应用开辟新的途径，为开发利用盐碱地和沿海滩涂，都具有重要的意义。