Isolation And Expression Analysis Of The DREB1Transcription Factors From Chicory And Establishment The Genetic Transformation System

Two novel DREB (dehydration responsive element binding protein) transcription factors, designated as CiDREB2A and CiDREB2B,were isolated from chicory. The temporal and spatial expression pattern analysis of CiDREBIA and CiDREB2B gene in different organs with or without external stress treatments were then carried out. Both subcellular localization assay and yeast one-hybrid system were carried out to study CiDREBs protein localization and transcription activity. We analyzed the effect of different phytohormone combination and concentration on the Puna chicory regeneration in vitro,and established the genetic trasformation system in Puna chicory. This study provides the investigation of Cichorium intybus abiotic tolerance mechnism, moreover, this study exploites stress tolerance genes from Cichorium intybus and new breeding methods. The main results were described as followed:1. Based on electronic cloning method of bioinformation and known DREB1from other plants,two assembled EST with ORF were obtained. Using specific primers,we isolated two transcription factors, CiDREBIA (KC801338) and CiDREB1B (KC811151). Their ORF were699bp and669bp, encoding232and222amino acids,respectively. Bioinformatic analysis showed that CiDREB1A and CiDREB1B had a conserved AP2/EREBP domain and a nuclear localization signal. Sequences of CiDREBIA and CiDREB1B shared higher similarity with DgDREBIA and DgDREBIB genes from chrysanthemum. The temporal and spatial expression pattern analysis confirmed that CiDREBIA and CiDREB1B had relatively higher expression levels at the young stage of chicory, after that the expression levels gradually decreased. The expression pattern analysises of CiDREBIA and CiDREB1B under different abiotic stresses revealed that they were induced significantly by cold and drought.This showed that CiDREB1A and CiDREB1B participate in the transcriptional regulation of chicory under the low temperature stress.2. Fusion vector CiDREB1A-GFP and CiDREB1B-GFP were successfully transformed into onion skin cells by gene gun method.The result of transient expression showed by the confocal scanning microscope indicated that the CiDREB1A and CiDREB1B gene products were located in the nucleus. Yeast one-hybrid system showed that yeast transformed with vector of pGADT7-AD-CiDREB1A and pGADT7-AD-CiDREB1B could grow on the selective medium (10mmol/L3-AT+SD/-His).This result indicated that the AP2/EREBP domain of CiDREB1A and CiDREB1B had the specific binding ability with DRE Cis-acting Element.Therefore,it confirmed that CiDREBlA and CiDREB1B had the fuction as DREB transcription factors.3. Effect of hormone type on the Puna chicory regeneration in vitro were studied and a reliabie and efficient regeneration system was established.The best regeneration medium for the Puna chicory was MS+1.5mg/L6-BA+0.2mg/L IBA.The regenerated shoots were transferred to1/2MS medium supplemented with0.1mg/L NAA for rooting. The optimal growth conditions would be:3days for pre-culture and co-culture with Agrobacterium tumefaciens of the explants; OD600=0.6and5minutes of infection time for infection conditions of Agrobacterium; Supplementation of100μmol/L acetosyringone to Agrobacterium co-culture medium and500mg/L Carbenicillin selected for disinfection. Resistant Puna plants were detected by PCR assay and the general genetic transformation efficiency of Puna chicory was10%according to our methods.