外源脱水应答转录因子DREB基因在转基因小麦中的诱导型表达与抗干旱生理效果研究

以冬小麦品种8901、5-98、99-92和104等品种的幼穗和幼胚为材料,用基因枪转化含逆境诱导转录因子DREB和bar基因的质粒pBAC128F（7024bp）。经筛选与植株再生,共获得70多个转基因小麦植株及其后代株系。转基因株系经PCR分析和RNA点杂交检测,结果表明外源转录因子DREB基因已稳定整合到转基因植株及其后代株系中,并且在部分后代株系中获得了表达。叶片脯氨酸含量测定表明,有16个转基因株系的脯氨酸含量与非转基因对照相比,增加相当显著,其中10个株系的脯氨酸含量在1100μg/g以上,比对照提高了2倍多。室内抗旱模拟实验表明,转基因株系停止浇水15d后,叶片仍然表现绿色,而对照叶片则失绿、枯干;复水10d后,转基因株系恢复活力,对照则死亡。研究表明,利用逆境诱导型启动子（rd29B）来增强外源DREB基因的表达,能显著改良小麦的抗旱性。     

Optimization of TaDREB3 gene expression in transgenic barley using cold‐inducible promoters

Constitutive over‐expression of the TaDREB3 gene in barley improved frost tolerance of transgenic plants at the vegetative stage of plant development, but leads to stunted phenotypes and 3‐ to 6‐week delays in flowering compared to control plants. In this work, two cold‐inducible promoters with contrasting properties, the WRKY71 gene promoter from rice and the Cor39 gene promoter from durum wheat, were applied to optimize expression of TaDREB3. The aim of the work was to increase plant frost tolerance and to decrease or prevent negative developmental phenotypes observed during constitutive expression of TaDREB3. The OsWRKY71 and TdCor39 promoters had low‐to‐moderate basal activity and were activated by cold treatment in leaves, stems and developing spikes of transgenic barley and rice. Expression of the TaDREB3 gene, driven by either of the tested promoters, led to a significant improvement in frost tolerance. The presence of the functional TaDREB3 protein in transgenic plants was confirmed by the detection of strong up‐regulation of cold‐responsive target genes. The OsWRKY71 promoter–driven TaDREB3 provides stronger activation of the same target genes than the TdCor39 promoter. Analysis of the development of transgenic plants in the absence of stress revealed small or no differences in plant characteristics and grain yield compared with wild‐type plants. The WRKY71–TaDREB3 promoter–transgene combination appears to be a promising tool for the enhancement of cold and frost tolerance in crop plants but field evaluation will be needed to confirm that negative development phenotypes have been controlled.     

酵母单杂交系统在植物抗渗透胁迫转录因子研究中的应用

酵母单杂交系统是由酵母双杂交系统衍生来的研究DNA与蛋白质之间相互作用的新型系统。该论文系统阐述了单杂交系统的基本原理和技术路线,详细综述了其在植物抗渗透胁迫转录因子各个研究领域的应用进展,即克隆抗渗透胁迫类转录因子基因,确定已知DNA-蛋白质的相互作用,定位已证实的具有相互作用的DNA结合结构域以及验证转录激活作用;并分析了当前该系统在植物抗渗透胁迫转录因子研究中存在的问题,进而结合自己的研究对解决问题的途径进行了探讨。     

基因枪法获得逆境诱导转录因子DREB1A转基因小麦的研究

以小麦品种H6756和藁城8901作为基因枪转化的靶材料,取其护颖至雌雄蕊原基形成期的幼穗,用含逆境诱导转录因子DREB1A和bar基因的质粒pAHC25轰击胚性愈伤组织,在分别含有5mgL和10mgLBasta溶液的培养基上进行筛选。得到的抗性愈伤组织在不含Basta溶液的培养基上再生培养,获得218棵再生植株。田间涂抹浓度为100mgL的Basta溶液检测后,对抗性植株作PCR检测,获得54棵再生植株。通过对其中20株T1代的PCR和Southern杂交分析,已获得14株含DREB1A和bar基因的转基因小麦植株,其中H675613株,藁城89011株。     

Analyses of abiotic stress and brassinosteroid-related some genes in barley roots grown under salinity stress and HBR treatments: Expression profiles and phylogeny

We aimed to investigate abiotic stress (WAK, HvPIP1.1, HvPIP1.2, HvPIP1.3, HvPIP1.5, CYCD3, DREB2) and brassinosteroid-related gene (DWARF4) expressions in barley (Hordeum vulgare L. cv. “Hilal”) roots grown under different salt (150 and 250 mM), HBR (0.5 and 1 μM), and salt + HBR applications during 48 and 72 h at dark with their controls. Phylogenetic trees were also constructed to observe relationships among genes found in other plants. The expression of HvPIP1.2 and WAK reduced after salt treatment while HvPIP1.3, DREB2 and DWARF4 expressions increased. HvPIP1.1, HvPIP1.2, HvPIP1.3, HvPIP1.5 and DWARF4 expressions were upregulated under only HBR applications. Salt + HBR treatments increased HvPIP1.1, DREB2 and DWARF4 but decreased HvPIP1.2. Phylogenetic analyses indicated that Oryza sativa L. shared similar sequences with HvPIP1.5. CYCD3 could diverge relatively earlier from cyclin genes during evolution as it segregates in a distinct clade. Sorghum bicolor showed sequence homology with DREB2. Oryza australiensis L. and DWARF4 were found in the same clade. To our knowledge, this is the first detailed report related to salt stress and HBR applications in terms of the expression of different genes in barley, providing a valuable information for molecular breeding improvement of stress-related traits.     

DREB转录因子研究进展

DREB转录因子即干旱应答元件结合蛋白质,它能特异结合启动子中含有 DRE/CRT 顺式元件,激活许多逆境诱导基因的表达,增强植物对逆境的忍耐力。介绍DREB转录因子与DRE顺式作用元件的关系,DREB 转录因子与 DRE 元件的结合特异性,DREB 的结构特点和功能,DREB 转录因子的表达调控,DREB 转录因子的克隆及鉴定等方面的研究进展,简述 DREB 转录因子对调控逆境诱导基因的表达具有非常重要的作用,在提高植物综合抗逆性方面将有巨大的应用前景。同时,指出 DREB 转录因子在信号转导、作用机理及基因表达等方面的复杂性。      

Differential expression profiles of poplar MAP kinase kinases in response to abiotic stresses and plant hormones,and overexpression of PtMKK4 improves the drought tolerance of poplar

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules that play essential roles in plant growth, development and stress response. MAPK kinases (MAPKKs), which link MAPKs and MAPKK kinases (MAPKKKs), are integral in mediating various stress responses in plants. However, to date few data about the roles of poplar MAPKKs in stress signal transduction are available. In this study, we performed a systemic analysis of poplar MAPKK gene family expression profiles in response to several abiotic stresses and stress-associated hormones. Furthermore, Populus trichocarpa MAPKK4 (PtMKK4) was chosen for functional characterization. Transgenic analysis showed that overexpression of the PtMKK4 gene remarkably enhanced drought stress tolerance in the transgenic poplar plants. The PtMKK4-overexpressing plants also exhibited much lower levels of H₂O₂ and higher antioxidant enzyme activity after exposure to drought stress compared to the wide type lines. Besides, some drought marker genes including PtP5CS, PtSUS3, PtLTP3 and PtDREB8 exhibited higher expression levels in the transgenic lines than in the wide type under drought conditions. This study provided valuable information for understanding the putative functions of poplar MAPKKs involved in important signaling pathways under different stress conditions.