【作者】 王洋；

【导师】 王崇英；

【作者基本信息】 兰州大学 ， 细胞生物学， 2007， 硕士

【摘要】 高等植物生长发育的过程中经常会受到逆境胁迫，植物可以通过激活逆境信号传导途径表达特异蛋白产物，从而在一定程度上缓解胁迫压力。利用T-DNA插入诱发获得的拟南芥突变体155在表型上与野生型C24相比出现明显差异，课题组前期的测序结果显示突变基因与热激蛋白基因(Heat Shock Protein，HSP)101相关，是具有分子伴侣活性的ATP酶基因的结合亚单位，且突变基因的确对植株的高温耐受性起到作用。由于不同逆境信号途径之间存在广泛的交谈，本文试图在低温、干旱、高盐及极端光照强度等条件下研究突变体和野生型在逆境胁迫的敏感性和耐受度之间的差异，力图分析不同逆境信号之间存在的关联；并对突变体和野生型中作为渗透保护剂的脯氨酸和还原糖水平在正常生长条件下的差异和不同逆境胁迫下的变化趋势和程度进行了测定；同时对冷诱导途径中的主效转录激活因子CBF1进行了克隆和初步载体构建。获得了如下结果：1．野生型比突变体的冷抗性略强但不显著，且这种差异只出现在土壤材料中，而在培养基材料中观察不到野生型与突变体在冷抗性上有明显差异。无论野生型还是突变体，生长5周的材料比生长7周的材料具有更强的低温抗性，揭示了植物逆境抗性在其整个生命周期内是动态变化的。2．突变体在冷诱导条件下检测不到转录激活因子CBF1的表达，而野生型则表达强烈。由此，突变体似应表现出比野生型较弱的低温抗性，而实验结果并非如此。这可能是由于其他冷应答途径的补偿作用造成的，也说明了逆境信号途径的复杂性与多样性。3．对冷诱导过程中起主效作用的CBF信号途径的CBF1转录因子进行了基因克隆和初步的载体构建，期望可以通过基因工程手段获得具有优良低温抗性的转基因花卉品种。4．培养基盐浓度的逐渐升高对野生型和突变体的生长都表现出先促进后抑制的趋势。在较低水平下盐浓度升高对植株的生长起明显的促进作用，当盐浓度达到某个临界点后，再增加浓度反而对植株起抑制生长的作用。这说明，拟南芥存在最适生长盐浓度点(或区间)。在培养基材料上观察不到野生型和突变体在相同盐浓度下的差异反应。5．正常生长条件下，突变体中的脯氨酸和还原糖含量高于野生型。而在冷诱导或干旱胁迫条件下，突变体中脯氨酸和还原糖的积累总量均大于野生型，冷诱导7天过程中积累的脯氨酸总量大于干旱胁迫7天所积累的总量，说明低温比干旱更容易促进脯氨酸的积累；冷诱导条件下1至7天的脯氨酸积累量明显大于8至14天的积累量，说明脯氨酸积累呈现先快后慢的积累效应。突变体与野生型相比，在受到干旱胁迫时表现出更强的存活能力。这可能与正常生长条件下突变体中较高的渗透保护剂(如脯氨酸和还原糖)浓度相关。6．强光和黑暗条件影响了植株的正常生长，植株表现出叶柄变长，叶片黄化、卷曲，直到植株萎蔫的生理反应。极端光照条件对突变体的伤害略小于野生型。其原因有可能是野生型和突变体在信号传导途径上的差异表达，也可能是突变体较小的叶片面积和蒸腾作用。在野生型和突变体中，低温、干旱、强光、黑暗等胁迫条件都共同表现出延迟开花的效应，并且黑暗条件比强光条件造成的延迟更严重。以上研究结果说明，155突变体与野生型相比在多种胁迫条件下表现出不同的敏感性和耐受度，以上的差异是由T-DNA插入导致相应基因功能丧失而造成的。由此，我们推断在拟南芥热激反应信号途径和其他逆境信号途径之间存在某种形式的交谈，当突变体中HSP101基因表达阻断时，CBF1基因的表达也受到影响，植株的高温和低温耐受性均低于野生型。而突变体中渗透调节剂(脯氨酸和还原糖)的含量则显著高于野生型，且胁迫条件下积累量更多，干旱胁迫时存活力更强。此外，突变体的极端光强耐受性也强于野生型。这说明植物不同逆境信号途径之间的交谈存在正向与方向，激活与抑制，补偿和阻断等多种方式，同时进一步印证了逆境信号途径网络的复杂性和多线性。更多还原

【Abstract】 Plants are usually facing the abiotic stresses during their growth and development. They can mitigate the stresses by activating special stress signal pathways to produce proteins with protective function. 155 mutant, which is obtained by T-DNA insertion, exhibits notable differences in phenotype comparing with wild type C24. Previously sequencing result indicates that the gene traped by T-DNA insertion may encode the protein related with the HSP101 (heat shock protein), which is a subunit of ATPase with the activity of molecular chaperon. Moreover, experimental result proved that the gene traped by T-DNA play a role in plant’s heat-resistance.Since there are the extensive crosstalks between different stress signal pathways, this thesis is mainly focused on the difference between mutant and wild type on the sensitivity and endurance of stresses, such as low temperature, drought, high salinity and extreme light intensity. We try to analyze the correlation between different stress signals, then measure the concentration and accumulating rate of osmoprotestants such as proline and deoxidize sugar under the normal condition and several stress pressure. The accumulating rate of mutant 155 is different from wild type C24. The results are as below:1. Although the difference in freezing tolerantce is not remarkable between mutant and wild type, wild type is more freezing-tolerant than mutant. Curiously, this difference only occurs in plants grow in soil, In contrast, plants grow in culture medium has no singnificance difference in freezing tolerantce between wild type and mutant. 5-weeks-old plants are more freezing-tolerant than 7-weeks-old plants both in wild type and mutant material, which may reflect the stress tolerance of plant, or at least the freezing tolerance is dynamic along the whole lifecycle.2. The expression of CBF1 in mutant after cold treatment (4°C, 2h) can not be inspected by RT-PCR, while the expression in wild type is clear and strong. From this result, it seems that the freezing tolerance of mutant should be much weaker than the wild type, but the experimental result is not. We suggested that the controversy may because of the compensation of other cold responsive pathways. Furthermore, it is also illustrate the complexity and diversity of the stress signal transductions.3. The gene clone of CBF1, the main transmission activator of the cold acclimation, and preparatory work of vector construction was done.4. The increase of the salinity of culture medium exhibits the tendancy of "promoting firstly and then suppressive"way. It means under the relatively low concentration, the increase of salinity promote the growth of both the mutant and wild type. When the salinity reaching a critical point, the growth of both wild type and mutant plants were suppressed. Obviously, there is an optimum salinity for plants, or at least, for Arabidopsis. We have not seen any observed different reaction between wild type and mutant on medium.5. The concentration of proline and deoxidize sugar is higher in mutant than in wild type under normal conditions. When plants are facing the cold acclimation and drought stress, the total amount of accumulation of proline and deoxidize is much higher in mutant than in wild type, higher when cold acclimated for 7 days than drought stressed 7 days, higher when cold acclimated for the first 7 days than the second 7 days. These results indicate that the accumulating rate of proline is decreased as time goes on, moreover, cold acclimation is more effective than drought stress to increase the content of proline. Mutant resists drought treatment longer than wild type. This observation is coincident with the higher concentration of osmoprotentant (for example, proline and deoxidize sugar) in mutant.6. Extreme light conditions such as high or dark affect the growth, plants exhibit physiological reactions such as prolonged petiole, etiolated and coiled leaves, withered stem and so on. Extreme light conditions hurt wild type more severely than mutant. The reason may be the differential expression of signal transduction between wild type and mutant, or the smaller leaves and transpiration of mutant. Stresses such as low temperature, drought, high and dark light condition delayed the flowering time in both wild type and mutant. Moreover, both mutant and wild type plants treated under dark conditions blossom later than high light treatment.All the results above illustrate that the notable difference on stress sensitivity and endurance between mutant and wild type are because of the loss-of function of mutated gene by T-DNA insertion. Therefore, we conclude that there is some kind of crosstalk between the signal pathways of heat shock reaction and other stresses. When the HSP101 gene is blocked in mutant, the expression of CBF1 is severely affected, the thermotolerance of mutant is weaker than wild type. Whereas the drought tolerance of mutant is stronger than wild type, for mutant possess higher content and accumulating rate of proline and deoxidize sugar. Moreover, the endurance of extreme light condition in mutant is better than wild type as well. These observations may prove the presumption that the crosstalk between different pathways is very complex and multi-linear.更多还原