拟南芥抗寒相关转录因子CBF下游基因的筛选以及低温对植物开花的影响

【作者】 赵奂；

【导师】 何奕昆；

【作者基本信息】 首都师范大学 ， 遗传学， 2007， 博士

【摘要】 寒冷是植物经常遇到的一种环境因素，植物在低温条件下有许多复杂的生理反应，其中，冷驯化作为植物在逆境条件下生存的保证显得尤其重要。我们的研究旨在发现植物冷驯化过程中受调控的基因，并探索一种基于拟南芥基因组全序列分析的转录因子下游基因筛选新方法。在研究过程中，我们发现植物从营养生长向生殖生长的转化在低温条件下会受到抑制，而这种抑制与春化途径相互独立。拟南芥通过冷驯化提高对低温的抵抗能力，CBF／DREB1是一个在冷驯化过程中起关键作用的转录因子家族，该转录因子操控很多抗逆相关基因的表达，是抗寒相关基因启动表达的总开关。研究CBF下游基因对分析冷驯化机制有重要的作用。我们试图通过CBF结合元件DRE的搜索筛选CBF下游基因。经软件分析，拟南芥基因组中有80个基因在启动子区含有DRE元件。利用基因芯片技术和RT-PCR技术对这些基因进行分析的结果显示，受CBF直接调控的基因占预测基因的16％以上，同时，许多低丰度基因得到了检测。该方法弥补了传统基因芯片方法的缺陷，因此一些以往用基因芯片技术无法发现CBF下游基因在本研究中得以验证。通过上述筛选试验，氨基氨酸渗透酶AtPPRC44被证明在CBF过表达植株以及低温处理植株中上调表达，说明低温条件可能通过CBF调控AtPPRC44基因的表达。氨基酸作为一种生物保护剂在植物抗逆过程中起关键作用。现已证明，CBF参与了调节脯氨酸低温条件下合成的代谢途径，但是合成后的氨基酸是如何在组织和细胞内部分配，AtPPRC44很可能给出这个问题的答案。目前，我们已对该基因进行了的组织和细胞水平的定位分析，并初步证明该基因可能具有氨基酸外向转运功能。人们对低温影响植物从营养生长向生殖生长转化的认识由来已久，春化的分子生物学机制一直是植物分子生物学研究的重点问题。低温作为春化的必要条件，常被认为是开花的孕育者。但是，本次研究结果显示，低温在打破对开花抑制的同时对开花也存在抑制作用。这一点体现在：开花时间随温度的降低不断推迟，开花途径整合因子Socl，Leafy，FT在低温条件下的表达受到抑制，CBF过表达植株开花时间推迟。通过对低温影响开花时间特点的分析我们认为，温度对开花延迟有累积效应，随温度的降低抑制开花的因子会增加，这使得低温对开花的影响比光周期和春化作用更复杂。CBF家族在一定温度条件下也参加抑花作用，对CBF1过表达植株的研究表明，CBF1通过抑制开花途径整合因子Socl和Leafy调控植物从营养生长向生殖生长的转化，CBF1过表达与光周期途径和春化途径是相互独立的。低温不利于植物的生殖生长。光周期和春化途径对开花时间调节的意义在于其对开花季节的选择，最终还是对开花时环境温度的选择。而低温这种环境因素是最直接的环境信号，植物开花时间对环境温度的适应比日照时间和春化更为重要。更多还原

【Abstract】 Cold is an environmental condition that plants always encounter. Plant physiological reactions are complex in cold. In these reactions, cold acclimation, which plays a key role to ensure plant exist in cold, seems very important. In this research, we aimed at finding more genes which were regulated by in cold acclimation and tried to explore a new way based on the search against whole genomic sequence to find the downstream genes of transcription factors. At the same time, we found that the transition from vegetative to reproductive development would be repressed in cold condition. The repression is independent of vernalization.The Arabidopsis was enhanced in resistance to low temperature by cold acclimation. CBF/DREB1, a small family of Arabidopsis transcription factors, plays an important role in resoponse to cold. It switches on the genes which can enhance the cold resistance in plant. So the study on the genes regulated by CBF/DREB1 is very useful to analyze the signal pathway and the regulation of gene expression in cold acclimation.We tried to find the downstream genes of CBF/DREB1 by searching its binding site DRE. 80 genes which contain DRE in promoter regions were found by the software, Element Finder, developed in this research. As a result, some CBF regulated genes, which have not been reported previously, were found and the method was proved to be useful in finding downstream genes controlled by a transcription factor.An amino acid permease AtPPRC44 was found to be up-regulated in cold condition and CBF1 overexpressed lines suggesting that the expression of the gene may be controlled by the CBF in cold. Amino acid which acts as a bio-antifreeze plays a very important role in plant resistance to cold. The proline concentration is higher in the plants cold-treated and CBF overexpression lines than that in normal plants. It was proved that CBF took part in the pathway of the production of proline. However, we don’t know how the amino acid was distributed. The gene AtPPRC44 may be the best answer to this question.The major developmental transition in plant is the switch from vegetative to reproductive development. The molecular base of vernalization is discussed frequently. Low temperature which is a key condition for vernalization was always regarded as the cradle for flower. But we found that cold not only broke the repression to the flower but also acted as a repressive factor to it, which can be proved by the repression of the floral integrator factor Soc1, Leafy, FT in cold, and the flowering delay in CBF overexpression lines. The repression to flowering was enhanced by gradually decreased temperature. This suggested that there may be a lot of genes taking part in the repression of flowering induced by cold. They were expressed one after another at different temperature, and their effect on flowering delay was accumulated. CBFs express and repress flowering at a certain temperature. CBF1 regulates flowering time through a vernalization and photo period independent pathway. It delays the transformation by the repression on Soc1 and Leafy.The Arabidopsis selects flowering season through photo period and vernalization pathway to finish the reproductive development in a suitable temperature. However, the temperature which acts as a direct factor may play more important role in regulate flowering than photo period and vernalization.更多还原