水稻鉈逆境下鉀離子競爭效應與轉錄體分析之研究

Effect of Potassium Competition on Thallium Accumulation and Transcriptomic Analysis of Rice under Thallium Stress

薛仲凱(Jhong-Kai Syue)

指導教授 ： 洪傳揚

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202100491

摘要

隨著鉈應用有關之科技興起，鉈對於生物體的毒害研究也逐漸受到重視。鉈對水稻之毒害機制研究仍相當少，其毒害機制與鉀競爭效應是本試驗之研究目的。水耕栽培幼苗試驗結果顯示，Kimura B水耕液中添加總鉀濃度10倍之不同種鉀化合物和10 μM的硝酸鉈，其中不同鉀化合物包括KNO3、KH2PO4、K2SO4和KCl均能顯著抑制根部和地上部約70% 以上的鉈累積。全株水稻的水耕試驗中，在抽穗期處理相較於Kimura B水耕液總鉀濃度5倍或50倍KCl濃度於含1 μM鉈之水耕液中，均可顯著的抑制鉈在水稻葉片、莖部、根部、稻殼、穀粒的累積，在葉片、莖、稻殼可抑制約80% 以上，根部可抑制50%，穀粒則可抑制高達95% 以上，且不論5倍或50倍鉀離子影響效果皆相同，顯示鉀離子可有效地競爭鉈在水稻的吸收及累積。毒害機制方面，透過已知AOX過量表現轉殖株 (AOX-OE) 在鉈逆境下生長勢較野生型 (WT) 生長還要好，將AOX-OE和WT處理10 μM硝酸鉈24小時後，採集地上部做轉錄體分析。結果顯示24 H鉈處理會使泛素化作用發生，抑制光合作用相關蛋白之表現，並誘導穀胱甘肽還原酶 (GR3) 和抗壞血酸過氧化酶 (APX7) 等抗氧化酵素表現，而AOX-OE在鉈逆境下表現之穀胱甘肽轉移酶 (glutathione-S-transferase, GST) 基因種類和量都較WT還要來得多。荷爾蒙方面，不論AOX-OE或WT，鉈皆會抑制ABA生合成，並促使GA和乙烯生合成基因表現，且透過GA/ABA雙報導系統也能驗證該趨勢。高親和性鉀通道蛋白 (high‐affinity K+ transporter, HAK) 為重要之鉀離子吸收轉運蛋白，從其差異表現基因顯示，OsHAK5在AOX-OE中被誘導表現，而將三葉齡之AOX-OE和WT處理10 μM硝酸鉈一週，可以觀測到在AOX-OE根部鉈累積量顯著較WT根部還要來得少，因此這可能為AOX-OE較WT抗鉈逆境的原因之一，仍須進一步去驗證。從本試驗結果顯示，鉀會競爭鉈吸收通道進入水稻中，因此鉈可能透過鉀通道進入水稻中，同時，轉錄體分析結果顯示，鉈逆境的確會誘導GR3和APX7等抗樣化酵素的表現，而AOX-OE在鉈逆境下會誘導更多解毒重要酵素GST的表現，且AOX-OE本身會誘導OsHAK5表現，這些皆可能為AOX-OE較WT抗鉈逆境的原因。

關鍵字

鉈 ； 水稻 ； 交替氧化酶 ； 轉錄體分析 ； 鉀 ； 離子吸收 ； 累積

並列摘要

With the rise of technology related to thallium application, research on the toxicity of thallium to organisms has gradually noticed. Thallium toxicity study in rice has not been studied yet, therefore, this study aims to investigate the mechanism and potassium competition effect in rice. Hydroponic experiment results of rice seedlings showed that different minerals containing potassium including KNO3, KH2PO4, K2SO4 and KCl into half-strength Kimura B solution significantly inhibit up to 70% accumulation of thallium. At mature stage, 5 times or 50 times of potassium ion concentration treated with 1 μM thallium simultaneously in hydroponic solution during the heading stage can significantly inhibit the accumulation of thallium in leaves, stems, roots, husks, and brown rice. Tl accumulation of brown rice can be inhibited by more than 80% in leaves, stems, and husks, 50% in roots, and up to 95% in brown rice, and the effect of excess 5 times or 50 times potassium concentration is the same. The competition of potassium with thallium on absorption and accumulation in rice is present. In terms of the toxicity mechanism, AOX overexpression transgenic plant (AOX-OE) under thallium stress grows better than the wild type (WT) is known. AOX-OE and WT are treated with 10 μM Tl(I) for 24 hours and collected its RNA for transcriptome analysis. The results show that 24 H thallium treatment can cause ubiquitination, and inhibit the expression of photosynthesis-related genes, and induce the expression of antioxidant enzymes such as glutathione reductase (GR3) and ascorbate peroxidase (APX7). The type and amount of GST genes expressed under thallium in AOX-OE are much more than that of WT. In terms of hormones, thallium inhibits ABA biosynthesis and promotes the expression of GA and ethylene biosynthesis related genes. This trend can also be verified through the GA/ABA dual report transgenic rice system. High-affinity potassium transporter gene is an important for potassium ion uptake and transport. The differentially expressed genes (DEGs) show that OsHAK5 is induced by AOX-OE. 3-leaf-stage of OE and WT are treated with 10 μM thallium for a week, it can be observed that accumulation of thallium in roots of AOX-OE is significantly less than that of WT, therefore, this may be one of the reasons why AOX-OE is more resistant to thallium stress than WT, and further verification is needed. The results of this study show that absorption competition between potassium and thallium is present, and thallium stress does induce the expression of antioxidant enzymes such as GR3 and APX7. While under thallium stress, AOX-OE can induce more GST genes expression, and AOX-OE itself can induce OsHAK5 expression, which may be the reason why AOX-OE is more resistant to thallium stress than WT.

並列關鍵字

thallium ； Oryza sativa L. ； alternative oxidase ； transcriptomic analysis ； potassium ； ion uptake ； accumulation

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