Function Analysis Of GhABF1 And GhABF4 Transcription Factors Response To Stresses In Cotton

AREB/ABF transcription factor family was a very important component of ABA signal transduction pathway. The Sn RK2-AREB/ABF pathway controls most of ABA-induced ABRE-dependent gene expression in response to abiotic/biotic stress, usually in vegetative organs. In this study, ESTs of cotton AREB/ABFs were used as seed sequences, electronic cloning was use to combine into the full-length gene of Gh ABFs, containing the whole open reading frame(ORF), respectively. We designed specific primers according to the ORF, and cloned two Gh ABF transcription factor genes by PCR methods, designated as Gh ABF1 and Gh ABF4, respectively. To evaluate the two gene functions, the virus interference vectors, subcellular localization vectors, the prokaryotic expression vector and eukaryotic expression vector were respectively constructed. The charaterization and distribution of the two genes were analysis, and the interference plants and overexpression plants were treated with various stress. Colectelly, These results showed as below:1. Electronic cloning method was employed to combine into the full- length gene sequenes of Gh ABFs. Specific primers with enzyme sites were designed to carry out PCR using cotton c DNA as template. The ORF sequences of Gh ABF1 and Gh ABF4 genes were obtainedand respectively connected to the p GEM-T vector. The results of clone sequencing showed that two predicted genes were cloned successfully.The protein structure analysis revealed these two proteins belong to AREB/ABFs transcription factor family. The Gh ABF1 is 1281 bp in size encoding 426 amino acids, whose protein molecular weight and isoelectric point p I is 46377.1 Da and 9.79, respectively. The Gh ABF4 is 1227 bp encoding 408 amino acids. The molecular weight and p I of Gh ABF4 were 44277.7 and 8.88.2. Construtions of the interference expression vector of Gh ABF1 and Gh ABF4 were respectively designated as p YL156-Gh ABF1 and p YL156-Gh ABF4, which were transformed into Agrobacterium GV3101 by electric shock. The Agrobacterium harboring p YL156-Gh ABF1 or p YL156-Gh ABF4 was injected into the leaves of cotton seedings to silence the responding target genes. To evaluate the gene function, the Gh ABFs-silencing plants and the control were treated by the drought. The results showed that of the silencing plants is more sensitive to drought compared with the control.3. To analysis the subcellular localization of Gh ABF1 and Gh ABF4, p EGFPGh ABF1 and p EGFP-Gh ABF4 were developed and transformed into Agrobacterium GV3101 by electric shock. Agrobacterium was injected into the leaves of tobacco. And the epidermal cells of tobacco(Nicotiana benthamiana) leaves were observed under the fluorescent microscope two days after the injection. The results showed that p EGFP-Gh ABF1 fusion protein accumulated in nucleus, p EGFP-Gh ABF4 fusion protein accumulated predominantly in nucleus and was distributed on the cell membrane.4. The plant expression vector p Bin438-Gh ABF1 was constructed and transformed into Agrobacterium LBA4404 by electric shocks method. The transgenic tobacco plants were developed through Agrobacterium-mediated transformation method. The overexpression plants and the control were treated by drought and high salt stresses to analyze the functions. The resoults showed that the transgenic plants confered higher resistance to drought and salt stress than the control.