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Expression Analysis and Localization of OsDMI3 Gene Induced by Abscisic Acid

Author: SunLiLi
Tutor: JiangMingYi
School: Nanjing Agricultural College
Course: Cell Biology
Keywords: OsDMI3 ABA H2O2 Transient expression vector YFP Subcellular localization
CLC: S511
Type: Master's thesis
Year: 2009
Downloads: 31
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Abstract


Abscisic acid (ABA), one of the most important phytohormones, play an important roles not only to regulate many important aspects of plant-growth and development, but also to regulate plant adaptive responses to various adverse environmental conditions. Previous studies showed that one model of ABA action are related to plant oxidative stress signaling. ABA could cause the generation of hydrogen peroxide (H2O2) and enhance the capacity of antioxidant defense systems. Ca2+/CaM-dependent protein kinase(CBK) is Ser/Thr protein kinase. Ca2+/CaM- dependent protein kinases(CaMK) have been well researched in animals. Until now, many Ca2+/CaM-dependent protein kinases have been isolated from different plants. But the functions of most of CBKs in plants have not been clarified.Previous studies showed that CBK is likely to take part in the signal transduction of salt, cold, drought and Oxidative stress. Therefore, it is important to identify the relationship between ABA-induced oxidative stress signaling pathways and CBK gene to reveal the cellular and molecular mechanism of ABA signal transduction.In this study, we did something about that by using rice as model material and bioinformatics methods combine with molecular biology methods. An effort was made to elucidate the relationship between CBK genes(OsDMI3) and ABA, H2O2 in semi-quantitative PCR. Then we use the constructs (35S-pXZOsDMI3) and transient expression system to determine potential subcellular locations of OsDMI3. The results are as follows:1.100μmol·L-1 ABA treatment in the time-course of 4h showed that the transcript level of OsDMI3 in rice leves and roots reached maximum values in 30 min and 60min, then renduced to the control level. Through the treatment of ABA systhesis inhibitor fluridon(FLU), the results are that the transcript level of OsDMI3 in rice leves and roots was inhibited more significantly in the rice which were treated by ABA systhesis inhibitor fluridon(FLU) compared to the other rice which were not treated. However, the treatment of ABA largely removed this inhibition. These suggested that ABA can make the transcript level of OsDMI3 increasing.2.10 mmol·L-1 H2O2 treatment resulted in an analogical enhancement in the transcript level of OsDMI3 gene analyzed by RT-PCR. The time-course analysis of OsDMI3 gene expressions showed that the transcript level reached two maximum values in 4h of H2O2 treatment. To make sure that H2O2 took part in ABA signal transduction, pretreatments with H2O2 scavenger and inhibitor DMTU and DPI did not suppressed the increases of gene expression within 60min of ABA treatment. But the increases of gene expression within 90min of ABA treatment was significantly suppressed. These suggested that H2O2 is required for the ABA-induced up-regulation expressions of OsDMI3 gene in rice. A new evidence was provided to ABA-induced signal transduction in plant cell antioxidant protection system.3. To determine the location of OsDMI3 and through designing the primers which include restriction enzyme cutting site, the open reading frames were amplified. The gene were inserted into the pXZP008, respectively, under control of the CaMV35S promoter that allows the transient expression of protein in C-terminal fusion with YFP. The constructs (35S-pXZOsDMI3) were delivered into maize protoplast by PEG method. Images of OsDMI3-YFP fusion were captured with a confocal laser scanning microscope. OsDMI3-YFP were located to cytoplasm. This research prepares for further study about the relationship between ABA-induced OsDMI3 gene expression and protective antioxidant genes such as:Sod4, Cat1 and Apx1. The reorganized protoplast transient expression vector laid the foundation for understanding and expounding the network of ABA signal transduction pathway.

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CLC: > Agricultural Sciences > Crop > Cereal crops > Rice
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