【作者】 李丽娟；

【导师】 何奕昆；

【作者基本信息】 首都师范大学 ， 细胞生物学， 2005， 硕士

【摘要】 植物在生长发育过程中能感受干旱、高盐、寒冷等环境胁迫信号并通过一系列信号转导途径的作用诱导与抗逆相关功能蛋白的表达或调节酶的活性,以适应或抵御逆境胁迫。其中,转录因子对相关基因的表达起关键调控作用,并受信号转导途径的调节。在植物中存在着许多与胁迫相关的转录因子。DREB转录因子在干旱、高盐,及低温胁迫信号传递过程中起重要作用。该类转录因子特异存在于植物中,属于AP2/EREBP家族。一个DREB转录因子可以调控多个与植物干旱、高盐,及低温耐性有关的功能基因的表达。因此,利用转录因子来改良植物抗逆性,可以获得较为理想的综合效果,具有理论和实际意义。 小立碗藓(Physcomitrella patens)具有很强的抗旱、抗冷和抗盐胁迫的能力,体内存在丰富的高抗性基因资源。本实验利用RT-PCR,RACE的方法在小立碗藓中克隆了一个含有保守的AP2/EREBP DNA结合结构域的基因,命名为PpDREB-like。该基因属于AP2/EREBP家族转录因子,和ERF亚家族相比,该基因与DREB亚家族同源性更高。同时,PpDREB-like基因具有DREB亚家族成员所保守的V14和E19两个氨基酸,因此我们将该基因归属为DREB亚家族。对在胁迫处理下该基因的表达模式进行分析发现,PpDREB-like受干旱的快速和强烈诱导,在复水时,PpDREB-like基因的表达水平又下降到与正常水平几乎相同,在冷和高盐胁迫处理5hr时该基因也基本不受诱导表达。 我们构建了酵母重组质粒载体pREP5N+PpDREB-like和植物重组质粒载体pPZP+PpDREB-like,继而得到转基因酵母和烟草悬浮细胞,均表现出一定的抗性。这说明该基因转入细胞后,可能作为上游的调控因子启动了某些基因的表达,提高了它们的抗性。另外,我们还构建了一个植物反义表达载体,并将其转入烟草悬浮细胞,目前已经得到了愈伤组织,需要通过筛选和验证,进一步分析PpDREB-like基因的功能。更多还原

【Abstract】 The plants are greatly affected by environmental stresses such as freezing, drought, and salt. The plants can activate some proteins that are related to the tolerance or regulate the activity of enzymes by the signal pathway. The transcription factors play crucial roles in gene expression. There are some transcription factors related to abiotic stresses in plants.and they can be regulated by the signal transduction pathway. DREB proteins belong to AP2/EREBP transcription factors and are specific in plants. They are very important in mediating abiotic stresses such as cold, drought and high salt. DREB ptoteins can induce the expression of some target genes so that the plants can enhance the tolerance to drought and other stesses.Improving the tolerance of plants by using transcription factors has significance in theoretical and practical aspects.Physcomitrella patens has strong resistance to cold, drought and salty stresses and there are so many genes that can improve the tolerence of plants. Here, a gene is cloned by RT-PCR and RACE in physcomitrella patens It has a conserved AP2 domain and we named it PpDREB-like gene. The PpDREB-like gene belongs to AP2/EREBP transcription factors and the blast results show that it has more homology with DREB subfamily. In addition, it has conserved V14 and E19 of DREB subfamily. So the PpDREB-like gene is thought to be one of DREB subfamily. The results of RT-PCR under different stress conditions show that the expression of PpDREB-like can be induced by drought but not by redehydration and cold, high salt in 5 hours..Forthermore, the PpDREB-like gene was transformed into yeast and tabacoo suspension cell and its function is analyzed simply. Transgenic yeast and BY-2 cell exhibit certain resistance to osmotic stress compared with control. It indicates that the PpDREB-like protein can activte some target genes as a regulatory protein so that the cells can improve the torlerance to drought and other stresses. For further functional analysis on this gene, a anti-sense vector was constructed and transformed into BY-2 cell. The callus is being researched.更多还原