Production Of Transgenic Poplars With Anti-APX Gene And Study On Its Stress Tolerance

Poplar has a wide distribution and adaptable characteristic which plays an outstandingrole on vegetative building, soil and water conservation, forest industry and nitrogen fixation.Populus tomentosa is a special endemic populus of China. It is a heliophyte with a widerange of distribution, and is widely used as an important ecological protective forest andindustry forest. But Populus tomentosa is susceptible to a variety of adverse environmentalfactors, such as salt, drought and oxidation, etc. So it is extremely important to study themolecular mechanism of stress tolerance in poplars.Ascorbate peroxidase (APX) is one kind of important antioxidant enzyme in plants. Itcan remove H2O2by participating in ascorbate-glutathione cycle, especially clear the H2O2in chloroplast. APX(Mr35KD) is a peroxidase with protoporphyrin IX heme, containing theacid-alkali catalytic domain and heme-binding domain in its amino acid sequence. APX genefamily of plants contains cytoplasmic (cAPX), mitochondrial (mAPX), chloroplast (chlAPX)and peroxidase (mbAPX) four gene subfamilies. It often exists as a monomer, but there areseveral types cytoplasmic APX that can form dimers. Populus tomentosa741was used asexperimental material in this paper to produce transgenic poplars with antisense APX-IV geneby Agrobacterium-mediated transformation. At the same time, we studied the action ofAPX-IV gene on the transgenic poplars tolerance to salt, drought and oxidant stresses. Theresults are as follows:1Mediated by Agrobacterium containing anti-APX-IV gene recombinant expressionvector to transform poplar, we screened the adventitious buds and adventitious roots underselection pressure of20and10mg/L Kan respectively, and ultimately won the regenerationplants resistant to Kan.2Extracting the total DNA from the above transgenic plants preliminarily obtained,using the primers designed from selective marker gene npt-II, the plants which could beamplified the objective strap about700bp by PCR were the positive plants. The PCR results revealed that we have successfully obtianed seven strains of transgenic poplars with antisenseAPX-IV gene.3Under normal conditions, the transgenic poplars above had a longer growth cycle, lessand thinner roots, compared to the wild type.4Under Salt conditions, the plants with anti-APX-IV showed some symptoms of saltinjury earlier than wild-type plants. After4days treated with NaCl, the transgenic plantsshowed blade tip yellow and scorch phenomenons appeared on part of the leaves. Thedetermination results showed that the increased amplitudes of APX and GR activities intransgenic plants were lower than the wild type, and the APX activity value of the former wasrelatively low. The lower activity of antioxidant enzymes made transgenic plants accumulatemore reactive oxygen species which led to the ascending range of relative conductivity, MDAcontent and accumulation of H2O2greater at the same time. The AsA content, chlorophyll a, band total chlorophyll content in the transgenic plants, however, declined rapidly.5Under drought condition, transgenic Populus tomentosa showed injured symptomson leaves such as curl and wrinkling, edge necrosis and so on earlier than the contrast. Onphysiological and biochemical aspects, the accumulation of MDA in transgenic poplars wassignificantly higher than the wild-type poplar, while APX and GR activities and AsAconcentrations were lower. These results suggested that the antisense expression of poplarAPX-IV gene reduced the transgenic poplars tolerance to drought stress.6After one day treated with oxidation from MV, the leaves surface of transgenic plantsbegan to appeare brown necrosis spots and damaged earlier than that of the wild type. All thephysiological and biochemical indexes determined in transgenic poplars revealed that theexpression of APX-IV gene was inhibited, thus caused the activities of APX and GR, contentof protein and chlorophyll were significantly decreased.