| Cotton is one of the most important economic crops in China. However, the yield and quality of cotton have been seriously influenced by the Verticillium wilt. Molecular biology has been employed to make cotton more resistant to this disease.Recent years, the ERFs (ethylene-responsive ERF factors) have been a hot pot in molecular mechanism of plant stress-tolerance and stress-tolerance breeding. The ERFs are involved in the responses to biotic and abiotic stresses, and play vital roles in stress signal transduction pathways. Many studies have showed that the ERFs can activate pathogenesis-related (PR) genes by binding the GCC-box cis-element in the pathogenesis-related (PR) genes'promoter. This function is important to enhance plant resistance to pathogen. Therefore, the ERFs could be used to enhance the tolerance to biotic and abiotic stresses in crops.The aim of this study was to isolate and investigate new transcription factor genes related to stress tolerance, and then two ERF genes of Gossypium barbadense L had been isolated and charactered, and then dozens of transgenic plantlets had been obtained in this research.The following results were achieved through this study:1. Isolatlon of the ERF genes:In this study, two pathogen-inducible ERF (ethylene-responsive factor) genes in cotton (Gossypium barbadense L.) EREB1and EREB2, had been isolated and characterized. Analysis on the homology of amino acid sequences and the structure features of the 2 genes showed that EREB1 and EREB2 are different and new genes belong to the ERF subfamily. Southern blot results showed that EREB1 and EREB2 might be single copy genes in cotton genomic DNA.2. Analysis of the genes's expression pattern:The results of RT-PCR showed that the transcription of the EREB1 and EREB2 genes express in many organs and tissues, and were strongly induced by Verticillium wilt, especially in roots and stem of cotton. It was deduced that the transcription of EREB1 and EREB2 genes might play important roles in signal transduction pathways responded to the pathogen infection.3. Analysis of DNA-binding specificity:Electrophoretic mobility shift assays (EMSA) revealed that the ERF domain in EREB1 and EREB2 proteins could specifically bind to GCC-box cis-element, the result and these analysis further confirmed the DNA-binding specificity in vivo.4. Analysis of gene function:EREB1 and EREB2 were respectively transformed into Nicotiana babacum and Gossypium hirsutum L by Agrobacterium infiltration method, etc. Analysis of the genes's expression showed that the overexpression of EREB1 and EREB2 could enhance the PR genes in transgenic Nicotiana babacum.The overexpression of EREB1, and EREB2 can enhance resistance-related genes such as Chitinase ,β-1, 3-glucanase and PR genes in Gossypium hirsutum L.These results showed that overexpressing EREB1 and EREB2 genes could enhance the expression of downstream disease resistance related genes, the transgenic cotton were more resistant to pathogen compared with CK after infected by Verticillium wilt.In summary, these two novel ERF transcription factors genes from Gossypium barbadense L had the typical function of ERF. The EREB1/2 could activate the expression of pathogenesis-related genes, and then enhanced the pathogen resistance in the plantlets of transgenic tobacco and cotton. This research was the pilot work about the ERF genes in cotton(Gossypium hirsutum L), and further in-depth study was going on to test the overexpression of these two genes may enhance the resistance to pathogen, find more potential PR genes and lay the foundation for cultivating new pathogen resistant cotton lines through molecular breeding.
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