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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 is an important cash crop and its production and yield are affected by environmental stress such as drought, low temperature and high salt, so it is very important to increase the resistance of cotton to these stresses .DRE-dehydrationresponsive element binding protein DBP) plays a very important role in regulating the resistance of plants to environmental stress and the transgenic plants overexpressing DBP genes can resist these environmental stresses. Therefore, it is of great significance to study the effect of DRE element-binding proteins in cotton on cotton production It is very important.In the previous work, a DBP gene was isolated from cotton and named GhDBP1 and analyzed its expression characteristics in cotton plants at the transcriptional level.In the present study, the prokaryotic expression and purification of GhDBP1 Its DNA binding characteristics.The coding region of GhDBP1 gene was amplified by PCR and inserted into the prokaryotic expression vector pET28a and transformed into E.coli BL21 (DE3) .The GhDBP1 fusion protein was induced by IPTG in BL21 (DE3) Were successfully expressed.The Ni-NTA affinity chromatography technology was purified fusion protein.In the non-isotope gel retention experiments, pure Of the GhDBP1 fusion protein was able to bind to the DNA fragment containing the DRE element.In addition, the three-dimensional structure of the DNA binding region of the GhDBP1 protein was subjected to a computer simulation using the SWISS-MODEL software.The simulation results showed that the main DNA binding region of the GhDBP1 protein The structure of the strand structure and folding pattern are similar to that of the DNA binding domain of the known Arabidopsis GCC box binding protein AtERF 1. These results show that GhDBP1 is a dehydration response element (DRE) -bound transcription factor and may utilize a DNA sequence that is complementary to AtERF1 DNA A similar structure of the binding domain is combined with its target sequence dehydration response element (DRE).