Isolation And Characterization Of The Dreb Transcription Factor From Commander Chicory And Establishment Of Genetic Transformation System Of Commander Chicory

Abiotic stresses such as salt, drought, high-temperature and freezing caused severe damages on growth and development of plants, which are also the direct reasons of yield loss of crops. The adverse condition induced transcription factors which can regulate transcriptions of a series of downstream stress-responsive genes through their interactions with cis-elements to improve the integrated resistance of the plants. The DREB transcription factors of AP2family are the hot spot for the molecular breeding of transgenic tolerant plants in recent years. Cichorium intybus.L is a kind of plant resource as both medicine and food, which is also the main source of inulin. In the current, modern genetic engineering technology is a new way to chicory varieties breeding.In this study, three DREB-like genes were isolated and cloned from chicory and their functions were analyzed. At the same time we established agrobacterium mediated genetic transformation system for chicory to provide experiment basis for improving crop tolerance to harsh environmental using gene engineering. The major results were as follows:1. Three CiDREBs were cloned by cloning method in silico based on the EST sequences Chicory. To obtain the full length sequence of the gene, RACE experiments were performed. Sequence alignment showed that these genes possessed typical structure characteristics of AP2/EREBP domain and had high homology with other known DREB gene in the conservative domain. To investigate the expression pattern of DREB genes responsive to external stresses, total RNA were extracted and purified from chicory which subjected to various stress treatments (drought, high salt, low, high temperature and ABA) for different time. The results of RT-PCR analysis indicated that CiDREB genes may play important role in responsive to abiotic stresses in chicory. CiDREB2A could be induced by drought, salt, low temperature, high temperature and ABA. CiDREB5A could be induced by low temperature, high temperature and drought. CiDREB6A significantly induced by high temperature and drought but the expression profile was different in leaf and root. 2.Subcellular localization assay:In order to detect the subcellular localization of CiDREB transcription factor proteins, CiDREB2A、CiDREB5A、CiDREB6A were fused to the5’terminus of the coding region of enhanced green fluorescence protein(EGFP) under the control of the cauliflower mosaic virus (CaMV)35S promoter. Then the fused plasmids were introduced into onion epidermal cells using a particle bombardment method with a PDS1000/He. Transformed cells were incubated for12h at25℃in the dark and localization of the fusion proteins was then detected using a confocal microscope equipped with appropriate filter. The subcellular localization assay indicated that CiDREB2A、 CiDREB5A. CiDREB6A can localize into the nuclei. To investigate the DNA-binding specificity and activity in vivo, we fused the entire encoding region of DREBs into the yeast expression vector pGART7-AD and transformed into different yeast strains carrying different DRE elements. The result indicated that the yeast transformant with the DRE element was able to grow on selective medium lacking His, Trp and Leu in the presence of3-AT, whereas transformant with mutant DRE element was not able to grow. This indicated that CiDREB2、CiDREB5、CiDREB6were able to bind to DRE element specifically in yeast cells and activated reporter gene expression.3.Effects of culture medium composition on callus induction, shoot regeneration and root formation from cotyledon explants in commander chicory were evaluated to optimize the plant regeneration system. The Aeluropus littoralis Na+/H+antiporter (AINHX) gene, which encodes a Na+/H+antiporter, was introduced into chicory to evaluate the genetic transformation efficiency. The optimum medium for the callus induction and shoot regeneration of commander chicory were MS+1.0mg/L6-BA+0.5mg/L NAA. The optimum medium for commander chicory root formation was1/2MS+0.1mg/L NAA. The insertion of AINHX gene into chicory genome was confirmed by PCR. The genetic transformation efficiency of commander chicory was13.3%.The present study provides the basis to further study the molecular mechanism of the DREB transcription factors in regulating the stress networks in chicory, and good candidate gene resources for improving crop tolerance to adverse environmental conditions.