白菜型冬油菜抗冻蛋白的结构性质及其基因冷应激的分子机制

Cold and Freezing Disaster was one of the major abiotic stresses, resulting in a huge loss of agricultural production every year. Antifreeze proteins can be adsorbed to the surface of ice crystals. With modifying the morphology, inhibiting the growth and recrystallization of ice crystals, AFPs can reduce the freezing point of the solution. It plays a key role in plant resistance to freezing. In previous studies, antifreeze proteins have been isolated and purified from the super cold-resistant winter rapa after low temperature treatment. Based on the results, this project intends to analyze the physical and chemical analysis of BrAFP protein. BrAFP amino acid sequence was determined, and its active sites and functional domains were analyzed. The two level structure of BrAFP was determined by spectroscopy methods and bioinformatics methods respectively. Three level structure model of BrAFP will be constructed by homology modeling method. The specific affinity adsorption between BrAFP and ice crystals was simulated to clarify its molecular mechanism. Cloning of BrAFP gene, and analyzing its e transcription characteristics. Over expression vector will be construction to transformation plant. Changes of cold resistance of transgenic plant were identified. The aim is to reveal the molecular mechanism of BrAFP gene response to freezing.

低温冻害可造成作物生产极大损失。抗冻蛋白（antifreeze proteins,AFPs）能与冰晶特异吸附结合，在植物抗低温冷冻中发挥关键作用。前期已在低温处理的超强抗寒品种陇油7中，分离纯化出白菜型冬油菜抗冻蛋白BrAFP。本项目在此基础上拟对BrAFP蛋白质理化进行分析；测定BrAFP氨基序列并分析其活性位点和功能域特征；采用光谱学方法和生物信息学方法，综合确定BrAFP二级结构折叠形式及其比例范围；采用同源建模法构建BrAFP三级结构模型，对BrAFP-冰晶特异亲和吸附进行模拟，解析BrAFP-冰晶结合的分子机制。克隆BrAFP基因，分析其冷冻诱导转录表达特征；构建超量表达载体进行遗传转化，检测转BrAFP基因植系低温转录水平和BrAFP蛋白活性，鉴定转基因植株抗冻性变化，解析白菜型冬油菜BrAFP基因冷应激的分子机制。结果可为白菜型冬油菜抗寒机理的揭示和冬油菜分子育种奠定基础。

我国北方旱寒区存在大量冬闲田，春季裸露农田风蚀扬尘造成诸多环境问题，冬油菜种植具有良好地表覆盖效果，旱寒区冬油菜种植的关键是品种的越冬抗寒性。抗冻蛋白能够与冰晶表面特异吸附结合，具有抑制晶核重结晶和冰晶生长活性，在防止植物机体冷冻结冰中发挥关键作用。本项目优化了双向电泳分离纯冬油菜蛋白质的技术条件；发现了低温处理后的白菜型冬油菜蛋白提取液具有很高的冰晶修饰和重结晶抑制活性，分离出两种候选抗冻蛋白：β-1,3-葡聚糖酶和类甜蛋白。利用碎冰亲和吸附法和SDS-PAGE法，分离获得38KD单一条带蛋白，具有明显冰晶形态修饰和晶核重结晶抑制活性，质谱鉴定该蛋白为β-1,3-葡聚糖酶，为白菜型冬油菜新的抗冻蛋白，命名为BrAFP1。克隆了白菜型冬油菜BrAFP1基因，明确了BrAFP1的序列同源性和分子进化特征。基于BrAFP1序列分析，明确了BrAFP1的理论理化性质和蛋白质结构。分子动力学模拟结果显示，将冰晶和BrAFP1分子置于水盒子中，BrAFP1与冰晶表面有较强的吸附结合能力，结合的主要作用力来自蛋白分子与冰晶间形成氢键，BrAFP1-冰晶分子可形成26个氢键，其中10个氢键是苏氨酸与晶格固定水间形成的。4℃~-4℃低温3h、18h~24h均可高效诱导BrAFP1在冬油菜叶中高表达，BrAFP1在叶柄和根中转录水平很低；亚细胞定位结果显示，BrAFP1主要在细胞核和细胞质中分布。构建了过表达载体，转化拟南芥获得BrAFP1的T3过表达株系，过表达BrAFP1可显著增强转基因株系抗寒性，而afp1突变体对变的冷更敏感，明确了BrAFP1在油菜抗寒中发挥的重要作用。克隆了14个不同抗寒白菜型冬油菜品种的BrAFP1基因，多序列比对表明BrAFP1的CDS区共有5处单碱基突变SNP，其中2处有义突变；相关性分析表明，第17位氨基酸变异与品种越冬率呈显著正相关（r=0.612*），且强抗寒冬油菜BrAFP1多肽链第17位更偏好络氨酸，而不是丝氨酸。细胞膜流动性改变和细胞骨架动态组装都参与了白菜型冬油菜BrAFP1的表达调控。