Over-expression Of TsCBF1 From Thellungiella Salsuginea Improves Maize Abiotic Stress Tolerance

Plant productivity is strongly influnced by abiotic stress conditions induced by drought, high salt and low temperature. Beside the traditional way of breeding, the great progress in plant gene engineering, along with the more and more new functional genes cloned, great ardour has been given to breed plants by genetic engineering. Transcription factors play important parts in regulating the expression of a series of stress response genes. So transforming a transcription factor is a more effective way to improve improving abiotic stress tolerance in plants.Lots of transcription factors regulating relevant gene expression of drought, high salt, low-temperature, hormone, pathogen reaction and development have been characterized and identified from high plants. Recently, DREB transcription factors have been identified in many plant species. They can specifically bind to the cis-acting element DRE/ CRT (core sequence is TACCGACAT), which can be found in the promoters of many genes concerning drought-, high salinity- or low-temperature tolerance. Over-expression of DREB/CBF in transgenic plants activates the expression of many genes involved in stress tolerance. Previous studies have showed that extrinisic CBF1 gene can improve abiotic stresses tolerance, but one of the problems is growth retardation. There is no report about TsCBF1 from Thellungiella salsuginea. This is the first report of transgenic maize heterologously expressed TsCBF1 and it has important sigality to investigate the abiotic stress tolerance and plant growth.In our assay, TsCBF1 was transformed into maize using Agrobacterium tumefaciens-mediated method. The transgenic plants of the T2 generation were identified by PCR and Southern blotting analysis. RT-PCR analysis showed leaves of independent transgenic plant lines had high expression levels of TsCBF1. While the expression levels of ZmDREB gene in transgenic maize were lower than that of wild type maize. They were both based on the expression level of the actin gene of the maize. And transgenic plants had the evident higher copies of DREB/CBF mRNA considered of the total DREB/CBF1 mRNA of TsCBF1 mRNA and ZmDREB mRNA.To study the changes in physiology and metabolism of T₂ generation lines, the transgenic lines and wild type plants were drought stressed before the blooming stage. In the experiment, four independent T₂ generation transgenic lines that didn't have evident phenotype differences with DH4866 were chosen. The seeds were planted in the same plots. Soil water content was maintained at 15-16%. The drought stress was lasted for 2 weeks before restoring watering the plants. Under normal growth conditions, no phenotypic changes could be observed in transgenic and wild type plants. However, over-expression of TsCBF1 gene in transgenic plants displayed an improved drought or salt tolerance compared with wild-type plants. This was demonstrated by plant height, branches of tassels , flowering-anthesis intervel, anthesis-silking interval, pollen viability, ear length, 100 grain weight and so on. The non-transgenic plants had less branches of tassels,,abnormal improvement of stamen, sterility of pollen,,long anthesis-silking interval, and less grain weight per plant, while the transgenic plants have improved drought tolerance,such as higher pollen viability and grain weight per plant.We designated the second day after the plants were drought-stressed, as the 0day, and samples were colleced at the 0, 1, 5, 10, 14 days after stress and the 7 days after restoring watering. And relevant water content, content of soluble carbohydrate, damage of cell membrane, MDA content, and proline in the leaves of the plants were investigated. We observed lower ion leakage, lower MDA content, higher relative water content of transgenic plants. Higher levels of content of soluble carbohydrate and praline were accumulated in transgenic maize than that in wild type after drought stress. These studies indicated transgenic plants could stay the normal metabolism by accumulated osmotic substance under drought stress condition.0.5%(W/V) NaCl was used to treat both the transgenic seedlings and wild seedlings, which were planted in the same pot,and the weight of seedling overground and underground were measured. The transgenic plants not only had higher salt tolerance but also grow normally. This may be because the transformed TsCBF1 gene hadn't activate the expression of the gene concerned plant growth.In a word, TsCBF1 gene have characteristic of DREB transcription factors and its expression in transgenic plants could improve tolerant to drought stress .The transgenic lines which overexpressed TsCBF1 gene can have improved stress tolerance and grow normally. These works laid foundation of using plant genetic engineering techniques to enhance abiotic stress tolerance in crops. Meanwhile, we can use the transgenic plants to further study the molecular mechanism of the transcription factor regulating the signal network of response to environmental stresses in plant, and provide a new gene to improve crop tolerance to adverse environmental conditions.