Isolation And Functional Analysis Of FeDREB1 And Its Promoter In Buckwheat (Fagopyrum Esculentum)

Buckwheat is a pseudo-cereal food crop which belongs to the Fagopyrum Mill of Polygonaceae. The main cultivars are tartary buckwheat(F.tartaricum Gaerth) and common buckwheat(F.esculentum Moench). Due to the short growth period, strong drought resistance and good adaptation to the environment, buckwheat has become a significant composition of the food crops in the arid and semi-arid areas of China. Moreover, buckwheat can serve as a conventional herb medicine as well. In recent years, with the enhancement of individuals' life quality, the demanding for buckwheat is growing. Hence, improving the stress-tolerant study of buckwheat on the molecular mechanism is of very importance to the genetic improvement of buckwheat varieties with high yield and excellent stress-resistant traits. To explore the responding molecular mechanism of buckwheat on stress resistance, the common buckwheat accession Xinong 9976 was chosen as the experimental material to isolate and identify the gene of the stress-resistance associated DREB transcription factor.The results in this study are as follows:(1) RACE cloning method was utilized to isolate a DREB transcription factor from the common buckwheat Xinong 9976. A CBF/DREB homologous gene, Fe DREB1, was isolated and identified. Protein sequence alignment and phylogenetic analyses grouped Fe DREB1 with the DREB(A-1) lineage. The length of this sequence is 859 bp, encoding a protein which is comprised of 189 amino acids. Protein Sequence analysis indicates that the Fe DREB1 protein possesses all the characteristics of the DREB(A-1) subfamily in plants, harbouring the nuclear localization signal, 1 AP2/EREBP domain, 1 DSAWR motif and 1 LWSY motif. Moreover, subcellular localization observations suggested that Fe DREB1 protein localizes in the nucleus. Yeast one-hybrid assays showed that Fe DREB1 protein specifically binds to the DRE sequence and could activate the expression of reporter genes in yeast. These results further suggested that the Fe RDEB1 protein was a DREB(A-1) transcription factor.(2) The plant expression vector p BI121-Fe DREB1 was constructed and introduced into the agrobacterium GV3101, then were transformed into Arabidopsis thaliana(Ecotype Columbia) using the floral-dip method. According to PCR and the real-time quantitative approaches, Homozygous T3 35S::Fe DREB1 transgenic plants and homozygous T3 wild type transgenic with the PBI121 empty vector(WT) were selected to analyze phenotype and drought-/freezing-resistance of 35S::Fe DREB1 Arabidopsis. Over-expression of the Fe DREB1 gene significantly enhanced drought and freezing tolerance of transgenic Arabidopsis, but resulted in growth retardation and delay of flowering. However, GA3-treated 35S::Fe DREB1 Arabidopsis can get back to the wild-type phenotype, and still exhibited a greater degree of freezing tolerance compared with the wild-type. Measurement of physiological parameters show that soluble sugar and free proline contents in transgenic plants were significantly higher than in WT plants with or without freezing or drought stress. Conversely, relative electrolyte leakages in 35S::Fe DREB1 transgenic plants following freezing or drought stress were lower than in freezing-/drought treated WT plants, whereas there were no significant differences in electrolyte leakage levels under normal conditions.(3) The seed germination experiment showed that the seed of 35S::Fe DREB1 Arabidopsis is highly sensitive to ABA application. Digital gene expression profiling(DGE) analysis indicated that there are 35 stress-resistance related genes being up-regulated significantly in the leaves of the 35S::Fe DREB1 Arabidopsis plant, within which 10 ABA-independent genes ERD7, ERD10, COR47, COR414-TM1, LEA14, COR15 A, RD29 A, P5CS2, At GOLS3 and COR15 B have been up-regulated dramatically. Meanwhile, a number of ABA-dependent resistant genes such as NCED3, AZF2, HAI2, RD29 B, ATDI21, TIP, RD26, RAS1, PEN1 and RAB18 have been up-regulated as well, revealing that the Fe DREB1 gene may participate in an ABA-dependent/-independent pathways.(4) q RT-PCR showed that no transcript of the Fe DREB1 was detected under normal conditions, while the Fe DREB1 could be induced by low-/high-temperature treatments, drought stress, and exogenous ABA application. Expression analysis further revealed that Transcript levels of Fe DREB1 in leaves and roots were obviously higher than stems under cold(4°C) and drought stress. Genome walking technology was utilized to isolate the Fe DREB1 promoter sequence from the common buckwheat accession Xinong 9976. Promoter sequence present that this 1710 bp promoter at least contains 8 kinds of cis-elements which can correspond to the drought and low temperature response. To identify the-1710 bp upstream region of the Fe DREB1 gene required for promoter activity, we constructed a series of 5'-Fe DREB1 promoter deletion fragments. Each deletion construct was analyzed through Agrobacterium-mediated transient transformation in tobacco leaves treated with 4°C cold or drought stress. Promoter–beta-glucuronidase fusion assays revealed that the p CD1(-270 bp) deletion in the upstream region of Fe DREB1 could activate expression of the GUS gene at 4°C. The p CD1(-270 bp), p CD2(-530 bp), and p CD3(-904 bp) deletion induced low-level GUS expression under drought stress. By contrast, p CD4(-1278bp) has shown to be more effective in this aspect due to its great enhancement to the GUS gene expression under the same condition.