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The Functional Study Of Thellungiella Halophila APX1 And APX4 Sun Nana

Posted on:2011-01-22Degree:MasterType:Thesis
Country:ChinaCandidate:N N SunFull Text:PDF
GTID:2120360308964907Subject:Developmental Biology
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Environmental stresses,such as high salt, drought, low temperature,leads to increased production of reactive oxygen species (ROS) in plant cells, ROS can cause extensive cell injury if they can not be scavenged efficiently. The growth, development and propagation of plant can be affected seriously by ROS. The Reactive oxygen species (ROS) include hydrogen peroxide (H2O2), hydroxyl radical (OH·), superoxide anion (O2-), singlet oxygen (1O2) and so on in plant cells. The scavenging systems of reactive oxygen species can be classified into two categories: enzymatic scavengers and nonenzymatic antioxidants in plants. Ascorbate peroxidase (APX) is an important enzyme of enzymatic scavenger system in plants. It is a heme-containing protein that plays a central role in scavenging H2O2 using ascorbate as an electron donor in higher plants.Thellungiella halophila, a close relative of the model plant Arabidopsis thaliana, has similar heredity features and growth habits with A. thaliana. At the gene level, T. halophila are predicted to be more than 90% identity with A. thaliana. T. halophila and A. thaliana both have the predominances of short life cycle, self-fetilization, small genome, abundant seeds, efficient transformation and mutagenesis. Therefore T. halophila becomes a new model plant for studying on the plant salt tolerance mechanism. This project is aimed to study the function of T. halophila ThAPX1 and ThAPX4 gene. The results were shown as follows:1. The ThAPX1 and ThAPX4 gene were obtained from ESTs (expressed sequence tags) acquired fromλZap-cDNA library of T. halophila in our lab. The full length of ThAPX1 is 1245 bp, and the protein encoded by this gene is constitutive of 250 amino acids; The full length of ThAPX4 is 1209bp, and the protein encoded by this gene is constitutive of 284 amino acids.2. The functions of ThAPX1 and ThAPX4 were studied by using the gene silencing of ThAPX1 and ThAPX4 in T. halophila.1)Gene silencing vectors of pCAMBIA3301:ThAPX1 and pCAMBIA3301:ThAPX4 have been constructed respectively, then we transferred the two ones into Agrobacterium tumefaciens GV3101.2)Using floral dipping method, the flowers of T. halophila have been transformed. And T0 transformants were screened by using Basta. Finally, we have gotten 20 Basta-resistant lines for the gene silence of ThAPX4.3)The molecular identification of these T. halophila Basta-resistant plants are as below: (1) PCR result showed that all transformants had strong positive signals that about 500bp of Bar gene was amplified, while no signal was shown in wild type plants.(2) The 20 Basta-resistance plants have been vertified using semi-quantitative RT-PCR to observe whether the ThAPX4 gene expressed in transgenic T. halophila. Comparing the expression level between transgenic lines and wide type by RT-PCR, we have obtained four lines where the level of RNA expression is weaker than that in wide type.
Keywords/Search Tags:Thellungiella halophila, gene silencing, APX1, APX4, Reverse Transcription-Polymerase Chain Reaction
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