【作者】 张玉娥；

【导师】 武维华； 薛勇彪；

【作者基本信息】 中国农业大学 ， 植物学， 2005， 博士

【摘要】 DOR((?)r(?)ught (?)esistance)是与金鱼草S-locus F-box(SLF)基因同源的拟南芥SLF超家族的成员AtSFL35。该基因的T-DNA插入突变可以导致拟南芥产生耐旱性。离体叶片失水实验、MS+甘油培养基实验和盆栽干旱实验都证明dor突变体和野生型相比表现为耐旱。dor的功能互补实验进一步验证了该突变体表型是由于DOR基因的T-DNA插入造成的,表明该基因编码一个耐旱性的负调控因子。定量-PCR结果证明DOR基因组成型表达,但从DOR::GUS转化实验看出,DOR基因在叶子的保卫细胞中特异表达,提示dor表现出的耐旱可能与气孔有关。因此,我们比较了野生型和突变体在正常情况下的气孔大小和干旱条件下KAT1和KAT2的表达,结果显示dor突变体由于相对含水量高而表现为耐旱。为了研究DOR基因突变后耐旱的分子机制,我们采用Northern杂交方法分析了与耐旱相关的基因DREB2A、RD29A、COR15A和RD22在野生型和突变体中的表达,结果表明dor突变体表现出的耐旱可能与这些基因在干旱胁迫条件下的表达升高有关,提示DOR基因在依赖ABA和不依赖ABA信号通路上都发挥作用。我们从转录水平还发现DOR基因可能也参与了抗寒途径。酵母共转化和pull-down实验证明了DOR在体内可以与ASK14和CULLINI蛋白形成SCF复合体。这些结果表明,在干旱条件下DOR作为耐旱负调控因子参与泛素/26S蛋白小体降解途径来抑制气孔关闭。更多还原

【Abstract】 DOR (DrOught Resistance) is AtSFL35, a member of Arabidopsis AtSFL (S-locus F-box-like) superfamily related to the Antirrhinum S-locus F-box gene(SLF). A T- DNA insertional mutant of DOR results in a significant increase in tolerance to drought. Detached leaves, MS plus glycerol and soil-grown plants all exhibit a drought- tolerant phenotype compared to wild-type plants. Functional complement action experiment showed that DOR mutation leads to the drought tolerance, suggesting that DOR encodes a novel drought tolerance repressor. Quantitative PCR showed that DOR expressed ubiquitously at a low level. Interestingly, GUS reporter gene studies indicated DOR is specifically expressed in guard cell, suggesting that the DOR may function in stomatal movement under drought stress. Furthermore, we compared the stomatal aperture under normal grown condition and KAT1 and KAT2 expression under drought stress between wild-type and dor mutant and revealed that the dor could limit its transpirational water loss in response to drought stress. To investigate the molecular mechanisms for the DOR action, we analysed the DREB2A, RD29A, COR15A and RD22 expression by RNA gel blot. The results showed that the dor mutant induced the expression of drought-responsive genes after PEG treatment, indicating that DOR may function in both ABA-dependent and ABA-independent pathways for stress-responsive gene expression. Meanwhile, we found that DOR also function in cold signal transduction. Yeast two-hybrid and pull-down assays showed that DOR protein is capable of interacting with ASK14 and CULLINl, suggesting that it could form an SCF (Skp1/Cullin or CDC53/F-box) complex. Taken together, these results suggest that the ubiqutin/26S proteasome-mediate proteolysis mediated by DOR acts a negative regulator of drought tolerance by inhibiting the stomatal closure under drought stress.更多还原