Prokaryotic Expression and Purification of a Cotton Dehydration Responsive Element Binding Protein GhDBP1 and Its DNA Binding Activity
HUANG Bo, LIU Jin-Yuan** (Laboratory of Molecular Biology and Protein Science Laboratory of the Ministry of Education, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China)
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 software. 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.
【Fund】： 国家重点基础研究发展规划资助项目(973)(2004CB117303);; 国家高技术研究发展计划(863)(2004AA222100 2002AA212051和2002AA207006)~~
【CateGory Index】： S562
【CateGory Index】： S562