Cloning And Genetic Transformation Of DREB2 Transcription Factor In Hordeum Brevisubulatum (Trin.) Link

With the development of society and economy, environment living beings depend on has had a huge change. High salinity,drought and low temperature have a serious adverse impact on growth and development of corps, economic plants and vegetation and adversely affect crop production, which is a global problem. One of the important restrictive factors to interfere growth of plant is changes temperature. Exploitation of novel botanical resources by bioengineering technology to promote the resist ability of crops to drought, high salinity and low temperature and then increase crop production not only is an important question related to the national economy and the people's livelihood, but also become a hot point of study of modern bioengineering technology and area of plant improving. Macroscopical ecological principle combines to the microcosmic genetic engineering technology have been a new area of research. But as a result of the complexity of abiotic stress reaction in heredity and physiology, the knowledge about molecular mechanism of the resistance to abiotic stress is far from necessity, which restrict the application of genetic engineering technique.Salt-stress caused by the salinization of soil is a main environmental factor restraining plant growth and yield, which severely restricts the development of modern agriculture. Studies on the molecular mechanism of plant salt- tolerant and cultivating new variety of plant resisting salt-stress are of great significance for developing and utilizing saline-alkali soil. Hordeum brevisubulatum (Trin) Link, an excellent forage with high nutritional value and yield, is of high performance great economic value. On the basis of previous researches on physiological and biochemical features of Hordeum brevisubulatum (Trin) Link and its salt-resistant mutant in our Lab, we probe into the salt-tolerant molecular mechanism of Hordeum brevisubulatum (Trin) Link, which is important for the further development of saline-alkali soil .DREBs transcription factors able to bind the DRE cis element in the promoter of some genes concerned with cold, drought and high-salt stresses, resulting in initiating the expression of these genes at transcriptional level. Therefore, it would be a good strategy to improve tolerance of various kinds of agriculturally important plants to abiotic stresses by CBF/DREB genes transfer. Using RACE methods, we obtained one cDNA clones encoding new DRE-binding proteins in Hordeum brevisubulatum, and named HbDREB2. It has a completed ORF. The HbDREB2 has 1128 bp in length and encodes a deduced protein of 264 amino acid residues. It is shown from Protein Blast data that these proteins belong to a typical member of the AP2/EREBP transcription family with conserved NLS"RKKKVRRR", AP2 DNA-binding domain and acidic region. These"signature sequences"clearly distinguish those proteins from other proteins that have the EREBP/AP2 domain. HbDREB2 is classified into the A-2 group of DREB subfamily based on multiple sequence alignment and phylogenetic characterization. We found that HbDREB2 is most closely related to the barley OsDREB2 (Oryza sativa, AF300971), showing 73 % sequence identity. Northern blot and RT-PCR analysis indicated that its expression induced by salt and weakly induced by drought, but did not respond to ABA and cold treatment. We further investigated the role of the HbDREB2 regulatory genes in salt acclimation in transgenic tobacco by gene transformation via Agrobacterium. PCR, PCR-Southern and RT-PCR analyses of transgenic plants showed that the HbDREB2 gene was integrated into the tobacco genome and expressed at the transcriptional level. In addition, we show that over-expression of HbDREB2 results in elevated levels of Pro compared with the wild-type plants. Taken together, our results indicate that HbDREB2 is a DRE-binding transcriptional activator, heterologous over-expression of HbDREB2 may confer salt resistance in transgenic tobacco plants. The HbDREB2-expression tobacco plants used in the experiments described here also display dwarf and a delay in flowering phenotypes.Analysis of mRNA accumulation profiles of HbDREB2 showed that the expression of it is induced by salt treatment, However, it is not induced by cold treatment. It was weakly induced by drought. Little changes in the amount of its mRNAs were observed during the process of ABA treatment. It is concluded that HbDREB2 would improve abiotic stress tolerance of plants through an ABA-independent pathway.