【作者】 李林辉；

【导师】 何奕昆；

【作者基本信息】 首都师范大学 ， 植物学， 2005， 博士

【摘要】 低温是影响植物生长、发育及地理分布的一个重要生态因子，限制了冷敏感作物在低温季节的种植，导致光合效率低下，作物产量降低。但是一些温带植物在秋末冬初感受逐渐下降的外界温度，体内发生一系列生理生化变化，产生了适应性反应，提高了对冬季低温的抵抗能力，表明植物为抵御低温进化出了一套完善的应答机制。深入研究植物冷适应机理对于农业生产如改良作物、增加冷季节的作物品种等意义重大。 小立碗藓（Physcomitrella patens）是最近几年发展起来的模式植物系统。其分布广、极耐逆境、易培养、培养周期短，容易进行遗传转化和同源重组。小立碗藓长期处于单倍体世代，对于遗传学研究来讲，单倍体植物基因突变或敲除所产生的形态或生理生化变化容易被监测到，这些使研究植物抗逆基因的功能极为方便。 本实验构建了一个小立碗藓配子体冷胁迫的SSHcDNA双向文库。在正、反向文库中，分别随机挑选了384个阳性克隆，进行斑点杂交。根据斑点杂交的信息，分别在正、反向文库中挑选了240个和180个克隆单向测序，各获得232条和162条EST。通过BLSTx和BLSTxn与GenBank的数据库比较，在正向文库的232条EST中，有215条EST具有编码蛋白质功能，其余17条EST序列与已知蛋白质同源性较低或找不到任何同源序列。在反向文库的162条EST中，有134条EST具有编码蛋白质功能，其余28条EST序列与已知蛋白质同源性较低或找不到任何同源序列。在正向文库的215条具有编码蛋白质功能的EST中，120条EST序列的功能已知，95条EST与推测、假想或功能未知的蛋白质具有很高的同源性；在反向文库的134条具有编码蛋白质功能的EST中，104条EST序列的功能已知，30条EST与推测、假想或功能未知的蛋白质具有很高的同源性。这些已知的功能基因分别来自32个（正向文库）和27个物种（反向文库）。正向文库的已知功能基因30.3％来自拟南芥（Arabidopsis thaliana）、15.8％来自水稻（Oryza sativa）、7.9％来自小麦（Triticum aestivum）、1.3％来自于小立碗藓（physcomitrella patens）；反向文库的已知功能基因19.8％来自拟南芥（Arabidopsis thaliana）、12.7％来自水稻（Oryza sativa）、11.6％来自于小立碗藓（Physcomitrella patens）、5.8％来自于烟草（Nicotiana tobacum. L）。对上述240条和180条EST片段进行重叠群分析分别有126条和106条非冗余EST，正向文库里有106条单一序列和20个重叠群；反向文库里有90条单一序列和16个重叠群。 对文库EST分析后表明，ABA、Ca²⁺、一些蛋白激酶参与了小立碗藓的冷胁迫信号转导过程；热激转录因子、AP2结合结构域转录因子、MYC相关的DNA结合蛋白、MYB家族转录因子、bZIP转录因子等转录因子参与了冷胁迫下下游基因的表达调控；离子通道蛋白、水通道蛋白、膜通道蛋白、Rab蛋白等参与了物质和更多还原

【Abstract】 Low temperature is considered one of the most important environmental factors limiting the growth, development, planting, crop productivity, photosynthetic rate, and distribution of plants. But some temperate zone plants can adapt the futurecoming low temperature by producing some chemical and physiological metabolin in end autumn and primary spring, thus improves the resistance to low temperature in winter. Indicating plants evolve a set of perfect mechanism to response for low temperature. A clear understanding of plant cold-induced mechanism is a significant breeding objective in agriculture for improving crop productivity, and much species can be planted in winter.Physcomitrella patens distributed in wide world is a new developing plant model system in recent years with advantages: enduring extremely condition; a short life cycle; easy to culture, genetic transformation and homologous recombination. It facilitates the understanding of gene function for its long haploidy generation.as its morphological, chemical and physiological changes resulted from gene mutation or knock out are easy to be identified.A two directional SSHcDNA library of cold acclimation about Physcomitrella patens gametophyte was constructed. Dot blot hybridization was prepared with 384 random positive clones from forward and reverse library separately by blotting on Hybond N~+ membrane. 240 and 180 clones from forward and reverse library were sequenced according to dot blot hybridization information; 232 ESTs in forward library and 162 ESTs in reverse library can be further analyzed separately. The sequence similarity analysis in GenBank by BLSTx and BLSTxn shows that 215 ESTs encode for proteins (92.7% of total 232), 17 ESTs had no matchs in 232 ESTs. 120 ESTs encode for functionally known proteins and 95 ESTs for hypothetical, putative and unknown proteins among 215 ESTs; and in reverse library, 134 ESTs encode for proteins (82.7% of total 162), 28 ESTs had no matchs. 104 ESTs encode for functionally known proteins and 30 ESTs for hypothetical, putative and unknown protein among 134 ESTs. These best matched protein sequences are from 32 species, 30.3% from Arabidopsis thaliana, 15.8% from Oryza sativa, 7.9% from Triticum aestivum, 1.3%from physcomitrella patens in forward library; but in reverse library, 19.8% from Arabidopsis thaliana, 12.7% from Oryza sativa, 11.6% from physcomitrella patens, 5.8% from Nicotiana tobacum. EST contig analysis shows that there are 126 nonredundant ESTs in forward library, out of 126 ESTs, 106 ESTs are更多还原