Molecular Cloning And Analysis Of GhERF7and GhERF8in Gossypium Hirsutum L.

AP2/EREBP family is one of the most important transcription factors in Eucaryote, and it plays avery important role in regulating plant growth, development and physiological processes. TheAP2/EREBP genes were always thought of the specific transcription factors in plant, but these were alsoisolated from yanobacterium (Trichodesmium erythraeum), ciliate (Tetrahymena thermophila) and virus(Bacteriophage Felix01and Enterbacteria phage RB49). The AP2/EREBP superfamily contains threefamilies by the AP2domain.The AP2family proteins contain two repeated AP2/ERF domains, the ERFfamily proteins contain a single AP2domain, and the RAV family proteins contain a B3domain and thesingle AP2/ERF domain. The ERF family was mainly divided into two subfamilies, ERF subgroup andCBF/DREB subgroup. After four ethylene-responsive element-binding proteins were firstly identified intobacca (Nicotiana tabacum), many ERF family genes were found in the different species. These ERFfamily genes were usually taken part in hormonal signal transduction, response to biotic and abioticstresses, and many metabolic regulations in plant species. In this study, two novel ERF genes wereisolated from Gossypium hirsutum L. Relative expression of two genes were measured by Real-timequantitative PCR (RT-PCR) at the different period leaves, tissues and hormone treatment leaves.Thenwe got the target genes sequences of ERF using the pull-down method. The study’s main results were asfollows:1. Based on NCBI, DFCI databases and the leaf cDNA library of our laboratory, two cotton ERFgenes, GhERF7and GhERF8were combined with splicing sequences and PCR amplification.2. The expression of GhERF7and GhERF8was different in radical, caulicle, spire, roots, stems,leaves and flowers in CCRI10, the highest expression level of the two genes were in the later stageof leaf maturation.3. During different stage leaves, chlorophyll content and the expression of ERF genes were measured.During the stages of leaf senescence, the expression levels of the two ERF genes showed thedifferent tendence with chlorophyll content, it indicated that GhERF7and GhERF8could haverelationship with leaf senescence.4. Cotton plants were treated with ethephon, jasmonic acid (JA) and abscisic acid (ABA). Underethephon and JA treatment, the expression of GhERF7and GhERF8were up-regulated in leaves,while there were no significant expressions under ABA treatment. The results indicated thatGhERF7and GhERF8could be in the signal transduction net of ethylene and JA, but not theABA-dependent pathway.5.1900bp promoter sequences of two genes were obtined using genome walking method.6. Using plasmid restructuring technology,6×His-GhERF7recombinant protein was obtained. Andthen the cis-acting element sequences of ERF were got as following the pull-down procedure.