Functional Analysis Of The Promoter Of PtCBF1, A CBF1-Like Gene From Poncirus Trifoliata(L.) Raf

Citrus, the most important fruit tree in southern China, is suffered great economic losses by freezing damage occurred frequently in many provinces. Creating cold resistant variety is the best way to solve the question of freezing injury. With the difficults of polyembryony, sexual incompatibility, a long juvenility and so on, the progress of citrus traditional cross breeding had been very slow. Plant genetic engineering provides a new opportunity for us to create cold resistant citrus. In the early studies, it was found that the development of transgenic plant was unnormal when the single foreign gene expressing was driven by constitutive promoters. Thus, environmental induciable promoters need to be cloned and studied, in order to get an effective promoter which in response to low temperature and also drives the related gene expressing better. The purpose of this study is to provide the foundation of understanding the mechanism of cold resistance in citrus.Based on the cloning of PtCBF1 promoter of Poncirus trifoliate (L.) Raf and the deletion fragments of promoter, the present study is to investigate the follow questions: whether the promoter is cold-induced promoter or not, find out the most active cold-sensitive promoter in these deletion fragments promoter and analyse the functional element of the promoter. Aimed at knowing more about the characteristics of the promoter.The main contents and results of research are as follows:1. According to the sequence of PtCBF1, the deletion fragment promoters were fused to GUS report gene, constructed the fused victor. Next, transferred this gene to tobbaco mediated by Agrobacterium.2. GUS histochemical staining and fluorescent quantitative analysis of the tested transgenic tobacco plants proved that the total length of promoter and 571bp promoter may have a highest activity; the PtCBF1 promoter may be cold-inducible; MYC elements may regulate the PtCBF1 promoter to respond low temperature signal and other element may regulate the promoter.3. RT-PCR result indicated that the PtCBF1 promoter may be not only a cold-inducible promoter, but also could be induced by the other stresses. 4. By the EMSA test, the MYC cis-acting element could bind protein and then induce the activity of promoter. But whether this cold-response regulation is similar to Arabidopsis thaliana or not is still to be further studied.