棉花脱水应答元件(DRE)结合蛋白GhDBP1的原核表达、纯化及其DNA结合活性

棉花是一种重要的经济作物,其生产和产量要受到干旱、低温和高盐等环境胁迫的影响,因此提高棉花对这些胁迫的抗性非常重要.脱水应答元件(DRE-dehydrationresponsiveelement)结合蛋白(DBP)在调节植物对环境胁迫的抗性中起到非常重要的作用.而且过量表达DBP类基因的转基因植株能够很好抵抗这些环境胁迫,所以研究棉花中此类DRE元件结合蛋白对棉花生产有非常重要的意义.在以前的工作中,从棉花中分离一个DBP基因,命名为GhDBP1并在转录水平上分析它在棉花植株中的表达特征.在研究中,报道了GhDBP1的原核表达、纯化和它的DNA结合特性.GhDBP1基因的编码区用PCR技术扩增出来插入到原核表达载体pET28a中,并转化到大肠杆菌菌株BL21(DE3)中.经过IPTG诱导,GhDBP1融合蛋白在BL21(DE3)菌株中成功进行表达.利用Ni-NTA亲和层析技术得到了纯化的融合蛋白.在非同位素的凝胶滞留实验中,纯化的GhDBP1融合蛋白能够结合到含有DRE元件的DNA片段上.另外,用SWISS-MODEL软件对GhDBP1蛋白的DNA结合区的三维结构进行了计算机模拟.模拟的结果显示,GhDBP1蛋白的DNA结合区的主链结构和折叠模式与已知的拟南芥GCC盒结合蛋白AtERF1的DNA结合区结构很相似.这些结果显示了GhDBP1是一个脱水应答元件(DRE)结合的转录因子,并可能运用与AtERF1的DNA结合区相似的结构和它的目标序列脱水应答元件(DRE)相结合.     

草坪草狗牙根中抗逆基因BeDREB的克隆及功能鉴定

DREB(dehydrationresponsiveelementbindingprotein)蛋白是一类在植物中所特有的,能与DRE(dehydrationresponsiveelement)顺式作用元件特异性结合的转录因子,调控与干旱、高盐以及低温等逆境胁迫应答有关基因的表达.根据狗牙根近缘植物的DREB转录因子的AP2EREBP保守结构域的基因序列,通过RTPCR和RACE的方法分别从冷诱导和盐诱导的狗牙根cDNA中扩增到了2个似DREB基因,分别命名为BeDREB1和BeDREB2,并已提交NCBIGenBank,其登录号分别为AY462117和AY462118.这两个基因的编码框均为753个碱基,编码251个氨基酸,具有DREB转录因子的典型特征.两种逆境胁迫下扩增的基因序列同源性很高,达到了97.8%.利用酵母单杂交真核转录激活的方法进行了功能鉴定,证明BeDREB1和BeDREB2蛋白均可以与DRE顺式作用元件结合,激活下游报告基因HIS3的表达.RTPCR结果显示,BeDREB1基因受冷胁迫诱导表达,而BeDREB2受盐胁迫的诱导表达,且随着诱导时间的不同,表达量也在发生变化.上述结果表明,从狗牙根中克隆到的BeDREB1和BeDREB2基因属于DREB转录因子家族的新成员,在狗牙根中分别与冷胁迫和盐胁迫的信号转导有关.     

玉米中与DRE元件结合的转录因子的克隆和结构分析

DREB类的转录因子特异性地与DRE元件（脱水应答元件）结合,在植物感受干旱,高盐及低温等逆境条件时,激活一系列下游逆境应答基因的表达。进一步的研究发现,拟南芥DREB蛋白的DNA结合域（AP2区）中14位的缬氨酸和19位的谷氨酸对该转灵因子与DNA结合起着关键性的作用。利用酵母单杂交的方法,我们从玉米（Zea mays L.)的cDNA文库中分离到一个编码与DRE元件结合的蛋白的基因,命名为maDREB1。酵母体内的反式激活实验表明,该基因编码和蛋白能特异地与DRE元件结合并能激活下游报告基因的表达。对maDREB1蛋白14位和19位的氨基酸进行单位突变和双点突变实验,发现14位的缬氨酸突变为丙氨酸后maDREB1几乎丧了其转录激活能力,而19位的谷氨酸突变为天门冬氨酸后maDREB1的转录激活能力也受到较大影响。     

银新杨中与DRE元件结合的转录因子的克隆及鉴定分析

DREB类转录因子特异地与DRE元件结合,在植物感受非生物逆境(干旱、高盐和低温)胁迫时,激活一系列逆境应答基因的表达。我们选用银新杨(Populus alba×P. alba var. pyramidalis)为材料,通过PCR和同源EST搜索的方法克隆得到了一个类DREB的基因,命名为PaDREB2。酵母单杂交实验表明,该基因编码的蛋白能特异地与DRE元件结合并激活下游报告基因的表达。用RT-PCR的方法研究了PaDREB2的表达模式,结果表明PaDREB2受低温、干旱和高盐的胁迫诱导。     

Determinants in the sequence specific binding of two plant transcription factors, CBF1 and NtERF2, to the DRE and GCC motifs

Arabidopsis ERF proteins such as DREB1, DREB2, and CBF1 bind to the dehydration-responsive element (DRE), which has the sequence TACCGACAT. Mutation analyses reveal that a central 5 bp CCGAC core of the DRE is the minimal sequence motif (designated as the DRE motif in this paper), to which the ERF domain fragment of CBF1 (CBF1-F) binds specifically with a binding K(d) at the nanomolar level. In contrast, the ERF domain fragment of the tobacco ERF2 (NtERF2-F) does not interact with the DRE motif, but restrictedly recognizes the sequence containing a minimal 6 bp GCCGCC motif (designated as the GCC motif in this paper). However, CBF1-F binds to the GCC motif with a binding activity similar to its binding activity for the DRE motif. These in vitro binding variations were further demonstrated through reporter cotransformation assays, suggesting that the DRE and GCC motifs are two similar sequence motifs sharing a common core region of CCGNC with a discriminating guanine base at the 5'-end of the GCC motif. Binding analyses with the mutated ERF domain show that such a unique binding of NtERF2-F to the GCC motif can be altered by the substitution of A14 with valine in beta-strand 2 of its ERF domain, the mutant NtERF2-F, ERFav, acquiring a binding to the DRE motif with a K(d) comparable to that for CBF1-F binding to the DRE motif. This demonstrates that A14 is an important determinant of the NtERF2-F binding specificity. A possible mechanism of the binding specificity determination is discussed.     

Characterization of the novel gene BjDREB1B encoding a DRE-binding transcription factor from Brassica juncea L

A novel DREB (dehydration responsive element binding protein) gene, designated BjDREB1B, was isolated from Brassica juncea L. BjDREB1B contains a conserved EREBP/AP2 domain and was classified into the A-1 subgroup of the DREB subfamily based on phylogenetic tree analysis. RT-PCR showed that BjDREB1B was induced by abiotic stresses and exogenous phytohormones, such as drought, salt, low temperature, heavy metals, abscisic acid, and salicylic acid. Gel shift assay revealed that BjDREB1B specifically bound to the DRE element in vitro. Yeast one-hybrid assay showed that full-length BjDREB1B or its C-terminal region functioned effectively as a trans-activator. Furthermore, overexpression of BjDREB1B in tobacco up-regulated the expression of NtERD10B, and BjDREB1B transgenic plants accumulated higher levels of proline than control plants under normal and saline conditions, together showing that BjDREB1B plays important roles in improving plant tolerance to drought and salinity.