| Diacylglycerol kinase (DGK), as the key enzyme, plays important roles in the generation of phosphatidic acid (PA). As a small molecular phosphatide on the membrane, PA has emerged as a new class of lipid mediators involved in diverse cellular functions in plants, animals, and microorganisms. It has been widely studied in animals, while the researches in plants are limited. In recent years, it is also reported that PA regulates many important aspects of plant growth, development, hormonal responses and signal transduction in response to the environmental stresses. Many biotic and abiotic stresses such as pathogen infection, drought, salinity, wounding, cold and so on triggers the accumulation of PA in plant in a fast minute. Compared with the researches on the PLD pathway, the PLC-DGK pathway was reported in a less proportion, especially in plant. In our study, we have over-expressed AtDGK7 in Arabidopsis. Genome PCR and semi-quantitative RT-PCR analysis were used to identify the positive transgenic plants. The T3 transgenic Arabidopsis were used to be conducted with stress resistance assay and relative physiological analysis. The expression patterns of genes relating to stresses were also detected. All of the results indicated that AtDGK7, as a plant subgroupⅡDGK member, was response to several environmental stresses stimuli and phytohormone. Over-expression of the AtDGK7 in Arabidopsis conferred the transgenic plants more tolerance to stresses. The main results are as follows:1. The AtDGK7 gene was coloned into pBI121 vector in a sense orientation and the over-expression transgenic Arabidopsis were generated with Agrobacterium-mediated transformation method. The genome PCR has identified 9 positive transgenic lines, five of which were conducted with semi-quantitive RT-PCR analysis. The AtDGK7 gene was expressed higher in the transgenic lines TG1, TG2 and TG4 while lower in TG3 and TG5. Consequently, the lines TG2, TG4 and TG5 were selected to be conducted with physiological analysis.2. The germination detection indicated that the over-expression AtDGK7 in transgenic Arabidopsis was not sensitive to ABA. Incubated in the 1/2 MS medium supplemented with 1μmol/L ABA, a majority of the transgenic Arabisopsis lines germinated and grew normally, while the germination and growth of the wild-type Arabidopsis were severely inhibited. Under the 200mmol/L NaCl conditions, the transgenic plants grew stronger than the wild-type plants, which indicated over-expression AtDGK7 plants may have conferred salt tolerance.3. When plants suffered from salt or drought stresses, some marker genes such as RD29A, P5CS1, P5CS2 were induced. In the transgenic Arabidosis lines, the expressions of P5CS1 and P5CS2 were higher than that in wild-type plants. But the expression of RD29A was not distinct. It was speculated that the transgenic plants may conducted the signal transduction via P5CS1 and P5CS2 mediation, and RD29A may not involved in this pathway. Besides, the expressions of ABI2 and HAB1 in transgenic lines were lower than that in wild-type plants. Accordingly, the over-expression AtDGK7 gene may have cut up the transcripts of P5CS1 and P5CS2, and the activities of protein phosphatase they encoded would also be influenced.4. Under the low temperature treatment, the transgenic plants grew much stronger than wild-type plants, and exhibited higher proline content, total SOD activities and lower MDA contents, which suggested that the transgenic plants suffered less injure under low temperature.5. Another important discovery in this research is that the flowering schedule of transgenic plants was ahead of wild-type plants in the same incubation conditions.
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