Study On ROS-Scavenging Gene Of Thellungiella Halophila

Ever since the introduction of O₂ into our atmosphere by O₂ -evolving photosynthetic organisms 2.7 billion years ago, reactive oxygen species (ROS) have been the unwelcome companions of aerobic life . In contrast to O₂ , these partially reduced or activated derivatives of oxygen [singlet oxygen (¹O₂), superoxide anion (O₂。- ), hydrogen peroxide (H₂O₂ ) and hydroxyl radical (OH- )] are highly reactive and toxic, and can lead to the oxidative destruction of cells. Consequently, the evolution of all aerobic organisms has been dependent on the development of efficient ROS-scavenging. In recent years, a new role for ROS has been identified: the control and regulation of biological processes, such as growth, cell cycle, programmed cell death, hormone signaling, biotic and abiotic stress responses and development . These studies extend our understanding of ROS and suggest a dual role for ROS in plant biology as both toxic byproducts of aerobic metabolism and key regulators of growth, development and defense pathways.Arabidopsis thaliana is always best model plant for the research of the plant molecular biology, but it is true glycophyte and can unclose less salt-tolerane mechanisms. Recently, Thellungiella halophila, a close-relative of Arabidopsis thaliana, which is been suggested and be developed as a new salt tolerance model plant. Thellungiella halophila is a salt-tolerance crucifer with high sequence similarity(about 90-95% on cDNA level) ,similar heredity characteristics and growth habits with Arabidopsis thaliana. However, Thellungiella halophila is able to withstand dramatic salinity shock up to 500mM NaCl. This plant does not produce salt glands or other complex morphological alterations either before or after salt adaptation. This suggests that the salt tolerance in salt cress results from mechanisms that are similar to those operating in glycophytes. Therefore, the study of difference regulation of Reactive oxygen gene network between Thellungiella halophila and arabidopsis is quite significant for expatiating plant response mechanism of oxidative stress, understanding the metabolism alteration and adjustment of plant under stresses and expounding the stress tolerance metabolism.1.Gene silence study of CSD2 and MDAR5 in Thellungiella halophilaRNAi of CSD2 and MDAR5 in Thellungiella halophila have been carried on by using the technique of post-transcriptional gene silence(PTGS), containing: RNAi of CSD2 and MDAR5 from Thellungiella halophila have been constructed; ThCSD2-pGSA1252 and ThMDAR5-pGSA1252 have transferred into Agrobacterium GV1101; Using Floral Dip method , the flower of Thellungiella halophila have been transformed and harvest a wealthy of transformed seeds; Screening the transformed seeds by using herbicide Finale and have gotten about 18 and 16asat-resistance plants of the gene silence of CSD2 and MDAR5 in Thellungiella halophila respectively; 7 of Basat-resistance plants in the gene silence of CSD2 and 10 of Basat-resistance plants in the gene silence of MDAR5 have been confirmed by PCR. The further molecular identification of these Basat-resistance plants are going on . The purpose of the project is to explore the salt-tolerance function and mechanisms of CSD2 and MDAR5 in Thellungiella halophila.2. Expression analysis of salt induce ROS-scavenging gene between Thellungiella halophila and Arabidopsis thalianaReal-time Quantitative PCR is a new technique with high sensitivity and high speed , which is applied to the research of gene expression analysis.The project have completed to analyze their expression model of more than 30 ROS-scavenging gene in both Thellungiella halophila and Arabidopsis thaliana by Real-time Quantitative PCR , and have gained some important experiment result. In Thellungiella halophila, the transcript level of more than half of the ROS-scavenging gene reached its top at 24h time point, while almost all of the ROS-scavenging gene which transcript level reached its top at 3-h time point are located in chloroplast.