Research Of Adonis CBF1 By SMART RACE And Realtime PCR

Chilling and drought are the main non-biological stresses which plant face. There are two major pathways during the processes of low temprature and dehydration responsion in higher plants. The one is ABA (abscisic acid) dependent pathway; the other is ABA independent pathway. CRT/DRE (C-repeat,dehydration-responsive element) and CBF1 (CBF/DRE-binding factor 1) are the important elements in latter.Many cold-regulated genes(COR)were isolated and characterized in Arabidopsis and other cold tolerant plant species.Studies on regulation of COR in Arabidopsis have resulted in the discovery of a family of transcriptional activators.of which,CBF1,a member of the gene family,controls expression of a battery of COR in Arabidopsis and other cold tolerant plant species.During recent years.CBF-like genes were found in the genomes of chilling-sensitive plant species such as tomato and maize. Over-expression of Arabidopsis CBF1 confers elevated tolerance to chilling and drought stresses in transgenic plants.These results promote our effort to identify and characterize CBF-like genes to improve tolerance of chilling sensitive plant species to chilling and drought stresses.Adonis amurensis Regel et Radd belongs to Ranunculaceae Adonis, it distributes only in Northeast China, Russa, Korea and Japan. Adonis amurensis always flower in early spring when most plants have no leaves. Usually, Adonis is used as a Chinese herb. Because it has strong heart, diuresis and calming influence.However, its special anti-chilling ability is never researched in China and abroad. Furthermore, there are only sequences of tRNA reported in GenBank. We noticed that the cold-tolerance Adonis is greater than plants around them. Based on the research of cold responsion, we suppsed that its high cold-tolerance is related to COR and transcription factors such as CBF1. We use SMART RACE (Rapid Amplification of cDNA Ends) strategy to get whole sequence of Adonis cDNA. SMART RACE is an easy and quick method which improved from regular RACE. With this method, we designed primers of CBF1 DBD and get Adonis CBF1 DBD. Then we designed Adonis CBF1 GSP based on Adonis CBF1 DBD. 3' end and 5' end of Adonis CBF1 cDNA were gotten by SMART RACE. Through bioinformatics software, the sequences of Adonis CBF1 DBD, 3' end and 5'end are spliced to a 648bp sequence. Analyze result shows that Adonis CBF1 is a 216 amino acid protein with threeβ-sheets and oneα- helix which is a tapical AP2 damain. Alignment result shows that AP2 domain of Adonis CBF1 and Arabidopsis CBF1 are 91% same. The differencial amino acids are not contacting residues, and they did not affect the structure. At the same time we quantitated mRNA of Adonis CBF1 by realtime PCR. Adonis CBF1 is approximate three folds to 24hours cold treated Arabidopsis at RNA level. High level of CBF1 expression is a possible reason of Adonis special cold resistence.