Overexpression Of PtADC Isolated From Poncirus Trifoliata Confers Dehydration Tolerance In Transgenic Tobacco

Polyamines are ubiquitous polycationic aliphatic compounds with biological activity and low molecular weight formed in the metabolic process, which play a protective role against stress in plant. Arginine decarboxylase (ADC) is one of the key enzymes in the synthesis of Polyamines, catalyzing arginine decarboxylation into Arg, then Arg hydrolyzing into putrescine.In this experiment, arginine decarboxylase gene from Poncitrus trifoliata (PtADC) was introduced into tobacco via leaf disk transformation method. T2 transgenic materials were treated for stress resistance analysis. The main results are as follows:1. PCR detection and semi-quantitative PCR analysis:12 putative transgenic tobacco seedlings were identified by PCR with both primers of ADCR+35SF and NPTII, followed by the obtaining of T2 transgenic materials. Two T2 transgenic lines were used for RT-PCR analysis, and the results revealed that the gene was expressed in tobacco.2. Putrescine's content determination:Two T2 transgenic line were selected for dehydration, and the stressed leaves were used for putrescine's content determination. Results showed that both before and after dehydration, putrescine's content in transgenic leaves was higher than the control's;3. Water loss analysis:detached in vitro leaves of 60 days transgenic seedlings and controls were placed in the room temperature for dehydration. During the course of experiment, detached leaves from the controls endured more severe wilted phenotype than the transgenic ones, and more relative water loss.4. Electrolyte leakage and leaf relative temperature detection:dlectrolyte leakage showed that no significant difference was observed between control and transgenic materials before dehydration, but significant higher in the former after dehydration. Relative temperature was higher in the transgenic plants both before and after dehydration.5. NBT and DAB staining:two staining experiment were performed to detect the reactive oxygen species in the stressed leaves. The color of transgenic leaves were similar with the control ones before dehydration but lighter after, suggesting that the transgenic leaves had less H2O2 and O2.6. Gene expression analysis:RT-PCR domenstrated that the expressions of APX, DREB, AMDC were higher in the transgenic materials both before and after dehydration.