Genetic Transformation Of BpERF1 And CabZIP And Preliminary Function Study Of BpERF1

Transcription factors play important roles in plants defense as they can activate the expression of relative defense genes through binding to the cis-acting elements locate at the promoter region of them. Isolation and the subsequently functional study of transcription factors is a major approach in the research of plants defense mechanism. In this paper, a BpERF(ethylene responsive element binding factors,ERF) and 3 CabZIPs(basic leucine zipper,bZIP) transcription factors which may involved in the stress resistance were isolated from cDNA library of Chinese cabbage (Brassica rapa subsp.pekinensis) and pepper(Capsicum annuum L.), respectively.The overexpression vectors of all the four genes were constructed and transformed into tobacco by Agrobacterium tumefaciems mediated method. The roles of BpERF1 in plants defense was further studied using the transgenic tobacco plants. The main result is as follows.1. A putative member of AP2/ERF transcription family was isolated from the cDNA library of Chinese cabbage leaves challenged with high temperature. The putative amino acid sequence analysis showed that it belongs to the ERF subfamily of the AP2 gene family. The predicted three dimension structure of the putative protein was consistent with the structure of AP2 domain of Arabidopsis thaliana AtERF1 in former study. What'more, the transient expression result showed that the BpERF1 bind specifically with the GCC box but not the DRE box. Thus, we assigned it BpERF1. The open reading frame of the BpERF1 was employed to construct the plants overexpression vector by the Gateway site specific recombination technology. The vector was transformed into tobacco subsequently by Agrobacterium tumefaciems mediated method, and the T0 transgenic line was acquired. The plants of T1 transgenic tobacco lines derived from the self cross of T0 transgenic tobacco were used for further functional analysis. Lots of T1 transgenic plants showed a lose of apical dominance. Results of the abiotic stress treatments such as osmotic, drought, high temperature and drought showed no significant diference between the T1 transgenic tobacco of BpERF1 and the wild type one. Results of pathogen inoculation experiment showed that the transgenic plants overexpressing BpERF1 were more susceptible to Pseudomonas.Syringae pv.tomato DC3000, Phytophthora capsici Leonian and tobacco mosaic virus when compared with the wild type ones. Thus, it was suggest that the BpERF1 might function as a repressor in the regulation of plants responsive to pathogen. 2. The induced synthesis of sesquiterpene class of phytoalexin functions greatly in the regulation of plants pathogen defense in solanaceae family. However, the molecular mechanism of it remains unknown. Our previous research showed that a G box which can interact with the bZIP transcription factor located at the promoter of pepper sesquiterpene gene. Thus,it is hypothesized that the bZIP transcription factors might play imporant roles in the induced synthesis of the sesquiterpene phytoalexin. In this study, several pairs of PCR primers were designed on the basis of the nucleotide sequence of three bZIP transcription factor of Capsicum annuum searched from the GenBank and the corresponding full cDNA were acquired from cDNA library of pepper leaves challenged with UV-B radiation, subsequently. Then, the overexpression vector, transgenic RNAi vector and vectors used for virus induced gene silence of all the three CabZIPs were constructed through the Gateway high throughout gene clone system. The overexpressin vectors were transformed into the wild type tobacco by Agrobacterium tumefaciems mediated method, and the T0 transgenic plants of 2 CabZIPs were acquired. And the effect of VIGS of the 3 CabZIPs on the disease resistance and effect of overexpression as well as silence of CabZIPs on transcription of sesquiterpene cyclase genes is now being carried out. The works laid the foundations for the further study of the regulation roles of the bZIP transcription factors both in the induced synthesis of phytoalexin and in plants pathogen defense.