The Effects Of APX Gene Antisense Expression On Stress Tolerance In Plants

Reactive oxygen species will accumulate in plants when they suffere in theenvironmental stress. If the ROS maintain in a high level and can not be removed intime, the plants will be injuried. Asecorbate peroxidase （APX） is one of the importantenzymes for plants in scavenging reactive oxygen, which mainly via the AsA-GSHcycle to clean the excess H₂O₂. Thus the ability of plants to resistance external stresscan be improved. In this paper, the conserved cDNA antisense sequences of apx genescloned from 741 poplar were designed and antisense expression vectors wereconstructed. Positive transgenic plantlets contained purpose gene fragments wereobtained. Affects of antisense express apx-I and apx-IX were analysed when tobaccosuffered in drought stress, salt stress and oxidative stress. The main results ofexperiment were showed as follows:1. Eight PBI121-apx antisense expression vectors were constructed andtransferred to Agrobacterium tumefacien LBA4404 respectively, and then weretransferred into Tobacco with leaf disc transformation. 20mg/L Kan was used as aselecte pressure to screen the adventitious bud. As a resulte, transgenic tobaccoresisted to kanamycin were obtained.2. In order to made a further test for the transgenic plantlets, tube plantles whichcould rooted in the selection medium were choosed for PCR detection and GUShistochemical staining. The resultes suggested that eight kinds of tobacco containedantisense expression apx were obtained successfully.3. Drought stress induced the leaves of transgenic plants wilting and yellowingearlier. The RWC of transgenic plants decreased more obviously than wild type.Antisense expression caused the APX activity lower in transgenic plants, and theyaccumulated more H₂O₂, the membrane permeability increased more significantly.The date indicated that antisense expressing apx-I and apx-IX increased the sensitivityof tobacco to drought stress. 4. Under salt stress, antisense expressing apx-I and apx-IX limited the APXactivity in transgenic plants. After two weeks salt treatment, the content of H₂O₂intransgenic plants was rised more significantly than non-transgenic tobacco. Relativeconductivity and MDA content increased obviously, while the chlorophyll contentreduced in transgenic plants. Compared with the wild type, antisense expressing apx-Iand apx-IX weakened the resistance of transgenic plants to salt stress.5. Compared with wild type, the APX activity of transgenic plants did not risesignificantly under oxidative stress induced by Methyl Viologen. But the SOD activityand H₂O₂content increased significantly. At the same time, the chlorophyll decreasedand MDA increased much more. It just to say, oxidative stress lead greater damage totransgenic plants than non-transgenic tobacco.