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Primary Study on the Function of Subunit c Genes of Vacuolar H~+-ATPase in Arabidopsis Thaliana by Overexpression

Author: SuJie
Tutor: LiGuoZuo；WangRuiGang
School: Inner Mongolia Agricultural University
Course: Biochemistry and Molecular Biology
Keywords: Arabidopsis thaliana VHA-c subunit Overexpression Light ABA Sugar
CLC: Q943
Type: Master's thesis
Year: 2009
Downloads: 105
Quote: 3
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Abstract


The plant vacuolar H+-ATPase (V-ATPase) is a membrane-bound transporter protein located on the tonoplast and various inner membranes. It uses the energy released during cleavage of theγ-phosphate of cytosolic ATP, thereby creating an electrochemical H+-gradient which is the driving force for a variety of transport events of metabolites. Under stress conditions, V-ATPase can adapt to various environments by auto-regulation. Hence, the V-ATPase was named the stress-responsive enzyme.V-ATPase of Arabidopsis contains 13 distinct subunits organized into two large subcomplexes: The cytosolic V1 and membrane V0. Subunit c were coded by five genes, are a major component of V0 sector. Also, many evidence indicated that subunit c is the most sensitive component when plants are subjected to stress conditions. In this dissertation, primary function of some VHA-c genes was analyzed by overexpression, and the results showed:1. The target gene expression level in the homozygous transgenic plants was detected by semi-quantitative RT-PCR. Totally, the corresponding transcript level in 2 lines out of 7 VHA-c1 transgenic homozygotes, 2 lines out of 6 VHA-c3 transgenic homozygotes, 7 lines out of 9 VHA-c4 transgenic homozygotes and 6 lines out of 7 VHA-c5 transgenic homozygotes increased dramatically, indicating that the overexpression constructs are effective.2. Phenotype analysis of the VHA-c5 transgenic Arabidopsis under normal light condition showed reduction in root length compared to the control. While grown under dark condition, the root length in 6 lines of VHA-c1, 5 lines of VHA-c3 and all lines of VHA-c4 tansgenic homozygotes were reduced significantly.3 With treatment by ABA, the relative root growth of 4 lines of VHA-c1 and 3 lines of VHA-c3 over-expressed homozygotes were longer than that of the control; And the extent of cotyledon unfolding were higher than that of the control; The germination rates of all lines of VHA-c1 and 5 lines of VHA-c3 over-expressed homozygotes are higher than those of the control.4 Under sugar stress, the germination rate of 5 lines of VHA-c1 transgenic homozygotes and 5 lines of VHA-c3 transgenic homozygotes on MS medium supplemented with various concentrations of glucose were higher than that of the control; and the germination rate of 4 lines of VHA-c4 transgenic homozygotes and all of VHA-c3 transgenic homozygotes on medium containing different concentrations of sucrose were higher than that of the control.The above results showed that ectopic expression of VHA-c1, VHA-c3, VHA-c4 and VHA-c5 affected the tansgenic Arabidopsis root cell expansion to some extent. In addition, over-expression of VHA-c1 and VHA-c3 resulted in loss sensitivity to inhibition by ABA in both root growth and seed germination, and also lost the sensitivity to inhibition by glucose and sucrose on seed germination. This indicates that the vacuolar H+-ATPase might be involved in the signalling pathway mediated by ABA and sugar.

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