| Summary: | 碩士 === 國立中興大學 === 生命科學系所 === 97 === The Arabidopsis AAA-type ATPase atSKD1 (suppressor of K+ transport growth defect 1) involves in protein trafficking, and atSKD1 knockout mutants lead to a lethal phenotype indicating atSKD1 is an essential gene in Arabidopsis. The deduced amino acid sequence shows it has high homology to mcSKD1 found in halophyte Mesembryanthemum crystallinum L. (ice plant). Furthermore, mcSKD1 is a salt-induced gene, and it has been suggested to play a role in salt tolerance in ice plant. In this thesis, I used transgenic Arabidopsis transformed with antisense atSKD1 or loss-of-function mcSKD1 to observe the changes in salt tolerance of Arabidopsis. The Arabidopsis whole-genome microarray biochip was used for analyzing the changes in gene expression to reveal functions of atSKD1.
Under normal growth conditions, the appearances of antisense atSKD1 transgenic T3 generations (named 4.1.7.7 .1, 4.1.7.7.2, and 4.1.7.7.3), and mcSKD1K177A transgenic T2 generations (named K177.8.1, K177.8.2, and K177.8.3) were similar to that of wild-type Arabidopsis. PCR amplification of T-DNA and GUS staining were used to confirm the insertion of T-DNA into chromosome. The exact T-DNA insertion site of 4.1.7.7 lines was identified by TAIL-PCR. The gene expression and protein accumulation of atSKD1 were reduced in 4.1.7.7 lines, while the K177 lines showed increased atSKD1 expression but no difference in protein accumulation. Next, I analyzed the ability of salt tolerance in wild-type and these transgenic plants, and found the root lengths of transgenic plants were shorter than those of wild-type plants under high salinity condition. Furthermore, the ratio of Na+/K+ in transgenic plants were higher than that of wild-type indicating the failure of maintenance of ion homeostasis in transgenic plants. The results suggested that reducing atSKD1 expression decreased the salt tolerance in Arabidopsis, and the alteration of atSKD1 enzymatic activity might affect the ability of atSKD1 to regulate ion homeostasis.
Changes in whole-genome gene expression in 4.1.7.7 and K177.8 lines (4.1.7.7.1 and K177.8.3) were analyzed by ATH1 microarray biochip. Compared with the gene expression of wild-type, the expression of 106 and 205 genes were significantly different in 4.1.7.7.1 and K177.8.3, respectively. After gene clustering, I found the gene expression of certain AAA-type ATPase genes were both up-regulated in 4.1.7.7.1 and K177.8.3, but none of them was classified in the same clade of AAA protein family with atSKD1. These AAA-type ATPase might not be functionally redundant to atSKD1. One of the significant difference AAA-type ATPase genes in 4.1.7.7.1, At2g47000 (PGP4), is an auxin efflux transporter gene suggesting that decrease of atSKD1 might affect auxin transport and as the result, elevate PGP4 gene expression. In addition, the expression of osmotic stress-induced genes, At4g27410 (RD26) and At3g17510 (CIPK1), were down-regulated. The results suggested that decrease in atSKD1 expression might indirectly affect stress-related genes and stress-tolerant ability in Arabidopsis.
In conclusion, atSKD1 involves in protein trafficking and has positive effects on Arabidopsis growth, development, and the maintenance of ion homeostasis under normal and stress conditions.
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