過量表現 HvICE1 或 AtICE1 ( Inducer of CBF Expression ) 水稻轉殖株之分子鑑定及非生物逆境耐受性分析

Abstract

植物處於低溫逆境可因冷馴化作用提升對低溫的耐受性。實驗證實 CBFs/DREBs ( C-repeat binding factor/ dehydration response element binding factor ) 為參與冷馴化過程之主要轉錄因子，且在單、雙子葉植物功能上都具有高度的保守性。近來於阿拉伯芥中發現 ICE1 ( inducer of CBF expression ) 為 bHLH ( basic helix-loop-helix ) 之轉錄因子，可作用於 CBFs 之啟動子調控該基因之表現。本論文旨在藉由水稻功能性基因體學之研究配合轉殖水稻之分析，瞭解阿拉伯芥 AtICE 及大麥 HvICE 在不同非生物逆境下所扮演之生理功能。同時比較 AtICE 與 HvICE 在非生物逆境下之作用方式有無不同。為達此目的，首先經由生物資訊方式，可知水稻基因體至少有 4 個 OsICEs。由水稻基因晶片之數據整理得知 OsICE1 於高鹽及乾旱處理下可被大量誘導，卻不受低溫影響；OsICE2 以及 OsICE3 之表現會受到高鹽及乾旱所抑制，而 OsICE4 則可被高鹽、乾旱處理所誘導基因表現，此外 OsICE4 和 OsICE3 皆可被低溫誘導表現。然後本試驗以台農 67 號 ( TNG67, 耐冷、耐鹽但不耐乾旱) 及台中在來 1 號 ( TCN1, 不耐冷、不耐鹽但較耐乾旱 ) 水稻進行不同非生物逆境處理下 OsDREBs 調控組 ( regulon ) 及相關下游基因表達模式之探討。結果發現 TCN1 於低溫處理時 OsICE2 表現量較 TNG67 為低，且OsDREB1F、OsDREB1G、OsDREB1H、OsDREB1I 及 OsDREB1J 於 TCN1 之表現量亦明顯低於 TNG67。 為了深入探討單、雙子葉植物之 ICEs 對非生物性逆境耐受性之影響是否相同，進一步建立 35S::AtICE1 及 35S::HvICE1 轉基因水稻。經南方墨點法鑑定轉殖基因插入拷貝數，並使用熱不對稱交錯聚合酵素鏈鎖反應方式取得插入基因之側翼序列，進行基因定型分析後，可確定轉殖株之轉入基因為單一或雙拷貝之同質結合體。RT-PCR 結果發現 35S::AtICE1 及 35S::HvICE1 轉殖株於正常生長條件下轉入之 ICE 皆可過量表現，但下游 OsDREBs 整體之基因表現量相較於低溫處理之非轉殖株而言並沒有相對大量提高。非生物逆境耐受性之生理分析，包括葉綠素含量、丙二醛及脯胺酸含量之測定顯示 35S::AtICE1 轉殖株其 OsDREB1A; 1B; 1C 及 2B 之表現量明顯增加者，可提高植株對低溫逆境之耐受性，但於高鹽及乾旱環境之耐受性並沒有明顯差異。然而，35S::HvICE1 轉殖株其 OsDREB1B、 1C 和 1E 表現量僅些微增加者之表現量，轉殖株可增加對乾旱及低溫的耐受性，但卻相對降低對高鹽逆境之耐受性。 此實驗結果表示 AtICE1 以及 HvICE1 之功能並不盡然完全相同，推測可能有其它 ICEs 參與 CBFs/DREBs 之調控或者 ICEs 需經複雜的轉譯後修飾作用，分別參與水稻於不同非生物逆境耐受性之反應。

Through cold acclimation, plant can increase its tolerance capability upon exposure to low temperature. Previous study showed that CBFs/DREBs (C-repeat binding factor/ dehydration response element binding factor) were the major transcription factors that involved in cold acclimation process. The function of CBFs/DREBs evolved highly conserve between dicot and monocot. Recent study from Arabidopsis has defined ICE1 (inducer of CBF expression), a bHLH (basic helix-loop-helix) protein, as an important transcriptional factor that acts on the promoter of CBF gene and regulates its expression. In this study, based on rice functional genomic approach with transgenic rice analysis, we aimed to understand physiological function of Arabidopsis AtICE1 and Barly HvICE1 under different abiotic stresses. Meanwhile, the action mold of AtICE1 and HvICE1 will be compared under various stresses. To reach this goal, first, by bioinformatics search at least four of OsICE genes were found in rice genome. From currently available rice microarray data revealed that OsICE1 expression was highly induced by salt and drought but not affected in low temperature. OsICE2 and OsICE3 transcripts were repressed upon exposure to salt and drought environment. On the other hand, OsICE4 expression was salt and drought induced. Besides, OsICE3 and OsICE4 gene expression were both increased under low temperature stress. Then, we used TNG67 (Oryza sativa L., japonica; cold and salt tolerant but drought sensitive) and TCN1 (Oryza sativa L., indica; cold and salt sensitive but drought resistant) rice cultivars to investigate OsICEs; OsDREBs and OsDREBs regulon-related downstream genes expression profiles under abiotic stress treatments. The results indicated that OsICE2 expression level were down-regulated quickly in TCN1 at low temperature. And the amount of OsDREB1F、OsDREB1G、OsDREB1H、OsDREB1I and OsDREB1J expressions in TCN1 were also less than those of TNG67. To further elucidate the physiological effects of AtICE1 and HvICE1 under various abiotic stresses, we generated ICEs-overexpressed transgenic rice lines, 35S::AtICE1 and 35S::HvICE1. By Southern blotting analysis, TAIL-PCR, and PCR-based genotyping, we determined the copy numbers of transgene, T-DNA inserted flanking sequence and obtained either one or two copies of homozygous transgenic lines. RT-PCR result showed under normal growth condition indeed we can detect the overexpression of ICE genes in 35S::AtICE1 and 35S:: HvICE1 transgenic rices. However; compared to low-temperature stress-treated wild type plant, the whole gene expression profile of ICE-corresponding downstream genes (OsDREBs) did not obviously changed. The physiological analysis of abiotic stress tolerance assay, including chlorophyll, malondialdehyde (MDA) and proline content measurements showed that 35S::AtICE1 transgene rice with OsDREB1A; 1B; 1C and 2B transcripts enhanced could increase cold tolerance but not for drought and salt tolerance. 35S::HvICE1 transgene rice that with slightly OsDREB 1B; 1C and 1E gene expression increased could raise up its drought and cold tolerance but not salt tolerance. Taken together, the above results suggested that AtICE1 and HvICE1 may function not exactly the same in cold acclimation pathway. This may due to other ICEs co-operate in the regulation of CBFs/DREBs and CBFs/DREBs regulon-related gene expression or ICEs activity can be adjusted through post-translation modifications that lead to different responses when exposure to different abiotic stresses.