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標題: 分析轉入反向atSKD1及功能缺失mcSKD1阿拉伯芥轉殖株之耐鹽生理特性及基因表現的改變
Analyses of salt tolerance and changes of gene expression in transgenic Arabidopsis carrying antisense atSKD1 or loss-of-function mcSKD1
作者: 何笠維
Ho, Li-Wei
關鍵字: atSKD1;鹽逆境;mcSKD1;salt stress;microarray;transgenic Arabidopsis;阿拉伯芥;微陣列生物晶片;耐鹽生理特性
出版社: 生命科學系所
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阿拉伯芥AAA-type ATPase atSKD1 (suppressor of K+ transport growth defect 1)已知參與細胞中的蛋白質運輸,並發現當atSKD1基因完全不表現時為致死之突變,表示在阿拉伯芥中atSKD1為一必要基因,又因在耐鹽植物冰花中之mcSKD1與阿拉伯芥atSKD1具有相當高的相似度,且mcSKD1為一鹽誘導基因,推測mcSKD1與冰花耐鹽性相關。因此在本論文中藉由觀察轉入反向atSKD1以及轉入功能缺失之冰花mcSKD1阿拉伯芥轉殖株,了解當atSKD1的表現量改變時阿拉伯芥之鹽耐受性是否改變,以及利用whole-genome生物晶片觀察受atSKD1基因影響後整體基因表現的變化,以期對atSKD1的作用以及基因表現影響的層面能有更深入的了解。
轉入antisense atSKD1之T3子代(稱為4.、、轉殖株與mcSKD1K177A之T2子代(稱為K177.8.1、K177.8.2、K177.8.3)轉殖株外表型與野生型阿拉伯芥並無明顯差異,利用PCR擴增轉殖株中T-DNA片段及GUS染色法確認T-DNA的插入,並利用TAIL-PCR定位4.1.7.7系列T-DNA插入位置。確認有T-DNA插入後觀察各轉殖株中atSKD1基因表現量及蛋白累積情形,發現4.1.7.7系列atSKD1基因表現量與蛋白累積情形皆有下降情形,而在K177系列部分atSKD1基因表現量增加但蛋白累積與野生型表現沒有太大差異。進一步針對各轉殖株進行耐鹽性之生理分析,發現在高鹽情況下各轉殖株根生長情況都較野生型差,並且植株中之Na+/K+比値也都高於野生型阿拉伯芥,說明轉殖株調節離子平衡能力較差。因此推測當atSKD1表現量下降會使阿拉伯芥對鹽耐受性降低,而轉入功能缺失之冰花mcSKD1則可能影響正常atSKD1功能而影響調節離子平衡之能力降低。
利用阿拉伯芥whole-genome生物晶片ATH1進行4.1.7.7及K177.8的子代(分別為4.及K177.8.3)基因表現變化之分析,其中4.發現有106個、在K177.8.3則有205個與野生型相比基因表現有顯著差異的基因,並將表現有顯著差異之基因進行分群後觀察,發現此二轉殖株中有數個AAA-type ATPase基因皆為表現量上升,但由於這些AAA-type ATPase在AAA蛋白家族分類中都與atSKD1屬於不同clade,因此推測可能與atSKD1的功能並無互補。其中4.有差異表現的AAA-type ATPase基因中有一At2g47000 (PGP4)為auxin efflux transporter基因,推測可能由於atSKD1表現量下降影響auxin運輸而使PGP4表現量提高。另外受滲透逆境誘導表現基因At4g27410 (RD26)及At3g17510 (CIPK1)也都觀察到表現量下降的情形,因此推測atSKD1表現量降低也會間接影響逆境相關基因,並可能影響阿拉伯芥對逆境之耐受能力。

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 .1,, and, 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 lines was identified by TAIL-PCR. The gene expression and protein accumulation of atSKD1 were reduced in 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 and K177.8 lines ( 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 and K177.8.3, respectively. After gene clustering, I found the gene expression of certain AAA-type ATPase genes were both up-regulated in 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, 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.
其他識別: U0005-1808200917444600
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