导　　师： 范云六

学科专业： 090102

授予学位： 博士

作　　者： ;

机构地区： 中国农业科学院

摘　　要： 干旱、盐渍和低温是诸多非生物逆境中对作物危害最为严重的自然灾害，它严重影响作物的产量和种植面积。因此，一直以来，人们都很注重对干旱、盐渍和低温等非生物逆境的研究。但由于非生物逆境的复杂性，人们对植物与非生物逆境之间的关系的理解仍比较有限，人们在改良作物抗非生物逆境方面的结果也不理想。随着分子生物学与现代生物技术的发展，使人们从分子水平上深入认识植物与非生物逆境之间的关系成为现实，并为改良作物的抗非生物逆境性能开拓了新的途径。 研究表明在干旱、盐渍和低温等逆境条件下，植物体内会产生大量的活性氧，形成氧化逆境。由于活性氧具有非常活泼的化学性质，它会对细胞造成严重的伤害，因此活性氧的及时清除对植物抗非生物逆境性能的提高具有重要意义。为了减小这种伤害，植物在进化过程中形成了酶类和非酶类的抗氧化系统。在活性氧的清除过程中，过氧化氢酶CAT1起作重要作用，但在逆境条件下，植物本身的抗氧化系统受诱导表达的能力比较弱，不能及时地清除体内的活性氧，影响了植物抗逆性的提高。在Cat1基因的启动子区含有ABRE顺式作用元件，它是逆境条件下Cat1基因响应ABA作用的重要元件，事实上许多抗非生物逆境相关基因的启动子区都含有ABRE顺式元件，所以通过ABRE 顺式元件来调控Cat1表达的转录因子的研究具有重要意义。因为这样的反式因子它不仅仅是能够调控Cat1基因的表达，还可能调控其它的一系列的抗氧化逆境基因和相关的抗非生物逆境基因的表达。因此，本论文开展了玉米Cat1基因ABRE顺式作用元件的反式作用因子的克隆和功能分析方面的研究工作。旨在了解逆境诱导Cat1基因表达的信号传导通路中的重要的未知调控因子：转录因子；并初步了解该转录因子在抗非生物逆境中的作用。 通过本论文的研究工作得到的主要结论如下： 1．构建了玉米17dpp、21dpp幼胚/(授粉后17天、21天/)cDNA文库，容量分别为5.2×10~6和3.34×10~6，文库重组率为85％，其中76％大于1kb。 2．采用酵母单杂交的方法对文库进行了筛选，共得到7个不同的克隆，命名为 ABRE结合蛋白（ABRE Binding Proteins，ABPs），分别是 ABPZ、ABP3、ABP4。 ABPS、ABP6、ABP7、ABPg。序列分析表明ABPZ、ABP4、ABP6、ABPg 属于bZIP类转录因子，ABP3、ABPS、ABP7属于bHLH类转录因子。它们 均含有保守的DNA结合结构域。实验证明所克隆的ABP在酵母体内均具 有ABRE结合特异性，这是玉米中具有ABRE结合特异性的转录因子的首 次报道。 3．根据ABPs的DNA序列推测出其蛋白序列并输入GenBank中，BLAST分 析表明：与ABPZ同源性最高的是拟南芥的ABF3，为34．l％；与ABP3同 源性最高的是水稻基因组中的SPAT为o6．9％＊ 但水稻中SPAT的研究尚未 有报道；与AB P4同源性最高的是水稻11LABI因子，为44．3％：与AB严 同源性最高的是拟南芥基因组中的 MYC蛋白，但同源性仅为 19％，且功能 未知；与ABP6同源性最高的是水稻基因组中的ABI，为54石％，但水稻中 ABI的研究尚未有报道；与ABP7同源性最高的是水稻基因组中的DNA结 合蛋白＊＊P，仅为26石％，且功能未知：与AB的同源性最高的也是水稻基 因组中的ABI，为41．9O。 4．ABP基因在盐、干旱、HpyABA、低温等不同逆境条件下的表达情况比 较复杂：盐仰aCI）可以抑制ABP4、ABP7的表达，诱导ABPZ、ABP工ABP工 ABPd、ABPg的表达。干旱诱导除ABP4、ABP7之外的所有的ABPs的表达。 H。O。抑帘ABP4、ABP7的表达，诱导ABPZ、ABP三、ABPS、ABP6、ABPg 的表达。ABA诱导除ABP4之外的所有的ABP的表达。低温对所有的ABP 都没有诱导作用。这种复杂性说明ABPs分别在不同的逆境条件下发挥着不 同的功能，它们既有区别又有联系，同时也反映出在逆境条件下植物细胞内 基因表达调控的复杂性。由上面的实验结果推知ABPZ、ABP3、ABP工ABP氏 ABP7、ABPg很可能是依赖于ABA的能够与Catl基因ABREZ顺式元件相 互作用的反式因子 CBF类；ABP4则很可能是不依赖于 ABA的能够与 Catl 基因ABREZ元件相互作用的反式因子CBFZ类。本论文的工作为非生物逆 境诱导Caf基因表达的信号传导通路的完善提供了新的数据。 5．通过5’RACE的方法得到了ABPZ基因的全长CDNA序列为1450hp，推测 的读码框为1056hp，编码351个氨基酸。ABPZ蛋白全序列与拟南芥ABRE 结合因子ABF3的同源性最高，但仅为33石％；ABP4基因的全长CDNA序 列为1835hp，推测的读码框为1083hp，编码360个氨基酸。ABP4蛋白与 水稻flMBI因子同源性最高，为44．3O，但在蛋白序列的N端ABP4比TRABI 且且 多出约60个氨基酸，ABPJ不受ABA的诱导，而TAsBI则受ABA的诱导， 看来二者的同源性虽然较高，但在功能上存在着差别； Plant productivity is strongly influenced by abiotic stress conditions induced by drought, high salt and low temperature etc.. Though people have paid attention to the research of abiotic stresses such as drought, high salt and low temperature for a long tune, poor understanding of the mechanism governing stress tolerance of plant remained because of the complexity of environmental stresses. So far, the effect of improving abiotic stresses tolerance in crops was not satisfied. The developments of molecular biology and biological technology offer new opportunities for understanding plant and abiotic stresses deeply and improving abiotic stresses tolerance hi plants. Plants produced lots of reactive oxygen species /(ROS/) and formed oxidation stresses under drought, high salt and low temperature etc.. ROS can react with a variety of biomolecules, altering or blocking their biological functions. It is very important to scavenge ROS hi time. To minimize the toxic effects of ROS, plants evolved highly regulated enzymatic and non-enzymatic mechanisms to keep a balance between ROS production and destruction. The antioxidant enzyme catalase is very important Among the enzymatic defenses. CAT1 of plants was thought to play an important role in antioxidant defenses, hi response to environmental as well as physiological stresses. The expression of antioxidant enzymes was induced weakly by plants itself, which was not enough to scavenge ROS rapidly. So, the studies on the transcription factors regulating expression ofCATl are important, hi this context, the cloning and functional analysis of trans-factors binding to cis-element ABRE2 of CAT1 gene in maize will be presented. The major conclusions are as follows: 1. 17dpp, 21dpp /(17, 21 days post-pollination/) cDNA libraries of maize embryo, containing 5.2 X 106 cfu and 3.34 X 106 cfu, respectively were constructed. The recombination rates were 85/%. The size of inserts was over 1kb. 2. The two libraries were screened by yeast one-hybrid system. Seven positive clones were named as ABPs /(ABRE binding proteins/). They were ABP2, ABP3, ABP4, ABPS, ABP6, ABP7 and ABP9. The results of sequence analysis showed that ABP2, ABP4, ABP6 and ABP9 belonged to bZIP transcription factor and ABPS, ABPS, ABP7 belonged to bHLH transcription factor. Seven ABPs mentioned above specifically bound to ABRE in yeast cells. 3. The analysis of ABPs protein sequences deduced in GenBank showed that the highest homology between ABP2 and ABF3 of Arabidopsis was 34.1/%; between ABP3 and SPAT of Oryza sativa was 36.9/%; between ABP4 and TRAB1 of Oryza saliva was 44.3/%; between ABPS and MYC protein of Arabidopsis was 19/%; between ABP6 and ABI of Arabidopsis was 54.6/%; between ABP7 and DBP of Oryza sativa was 26.6/%; between ABP9 and ABI of Arabidopsis was 41.9/%. 4. The expression of ABPs was induced by various abiotic stresses such as drought, high salt, low temperature, ABA, and H2O2. High salt induced expression ofABP2, ABP3, ABPS, ABP6 and ABP9 and inhibited that of ABP4 and ABP7. Drought induced expression of ABP2, ABP3, ABPS, ABP6 and ABP9 except for ABP4 and ABP7. H2O2 induced expression of ABP2, ABPS, ABPS, ABP6 and ABP9 and inhibited that ofABP4 mdABP7. ABA induced expression ofABP2, ABPS, ABPS, ABP6, ABP7 and ABP9 except for ABP4. Low temperature did not induce the expression of ABPs. So, ABP2, ABP3, ABPS, ABP6, ABP7 and ABP9 were likely to be the CBF1, which interacted with cw-element ABRE2 of Catl gene. ABP4 were likely to be the CBF2, which interacted with m-element ABRE2 of Catl gene. The above results offered new data for the signal transduction of Catl expression. 5. The full length of sequences ofABP2, ABP4 and ABP9 was obtained by 5'RACE. The cDNA sequence ofABP2 contained 1450bp and the open reading frame was 1056bp, encoding 351 amino acid. Compared ABP2 with ABF3 of Arabidopsis, the homology was only 33.6/%. The cDNA sequence of ABP4 contained 1835bp and the open reading frame was 1083bp, encoding 360 amino acid. Compared ABP4 with TRAB1 of Oryza sativa, the homology wa

关 键 词： 玉米 过氧化氢酶 转录因子

分 类 号： [S513.06]

领　　域： [农业科学]