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(英文)

Prokaryotic Expression and Purification of a Cotton Dehydration Responsive Element Binding Protein GhDBP1 and Its DNA Binding Activity

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.

Cotton （Gossypium hirsutum） is one of the most important economic crops in the world. Its growth and productivity were affected by environment stresses such as drought, cold and high salinity. Thus, the enhanced stress tolerance in this plant is of great importance. As the dehydration responsive element （DRE） binding protein （DBP） plays an important role in the regulation of plant resistance to environmental stresses and is quite useful for generating transgenic plants tolerant to these stresses, isolation and functional analysis of DBPs in cotton are important to cotton production. In the previous work, a DBP gene from cotton, named as GhDBP1, was isolated and its expression patterns in cotton plants was demonstrated at the transcriptional level. Here, the expression, purification and DNA binding activity of GhDBP1 were reported. The entire coding region of the GhDBP1 gene was inserted into an expression vector, pET28a, and transformed into Escherichia coli BL21 （DE3）. The fusion protein was successfully expressed under IPTG induction and the purified recombinant protein was obtained by Ni-NTA affinity chromatography. Non-radioactive electrophoretic mobility shift assay revealed that the purified GhDBP1 protein was able to form a specific complex with the previously characterized DRE element. In addition, the computer modeling of the DNA-binding domain of GhDBP1 were performed using SWISS-MODEL soRware. The main-chain structures and the folding patterns of the DNA-binding domain of GhDBP1 were similar to the known structure of the DNA-binding domain of the Arabidopsis thaliana GCC box-binding protein AtERF1. These results indicate that GhDBP1 is a DRE-binding transcription factor and might use the structure similar to that of AtERF1 to interact with DRE sequence.