水稻OsMAPK3基因於非生物性逆境及除草劑耐性之功能研究

Functional Characterization of OsMAPK3 Gene from Rice (Oryza sativa L.) Required for Abiotic Stress and Herbicide Tolerance

趙雲洋(Yun-Yang Chao)

指導教授 ： 劉麗飛

共同指導教授 ： 張孟基(Men-Chi Chang)

國立臺灣大學/生物資源暨農學院/農藝學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.02783

摘要

Mitogen-Activate Protein Kinase (MAPKs) 是參與植物生長發育、生物與非生物訊息傳遞逆境生理之重要一員，屬多基因家族。目前對各別MAPK 之生理功能及其如何參與調控生長發育或逆境耐受性之機制仍未明瞭。因此本論文首先探討 OsMAPK3 基因可能之功能，先就其核苷酸與氨基酸序列與其他水稻MAPK 基因進行同源性比對和演化圖譜分析後，並分析 OsMAPK3 基因啟動子上之順式反應 DNA 序列，結果發現OsMAPK3 與已知參與非生物逆境調控之OsMAPK4 同屬於C 群，在核酸序列上具有69％ 的相似性，但與其他 OsMAPKs 序列間的相似性僅在19％ ∼ 51％ 之間。此外 OsMAPK3 啟動子具有受環境、荷爾蒙及調控生長之序列，例如：DRE、HRE、CRE、ABRE、ARE及MYB等。表示OsMAPK3 可能參與諸多非生物性逆境之調控。當進一步以台農67號三葉齡水耕苗，進行不同逆境的處理，結果發現ABA 、NaCl、乾旱、H2O2 與 SA 可誘導OsMAPK3 基因之表現，但處理 paraquat、CuSO4、 CdCl2 和MJ 時則否。

為了深入瞭解OsMAPK3對水稻非生物性逆境耐受性之影響，本論文進一步以基因轉殖方法得到大量表現與抑制表現基因的轉殖水稻，並進行不同的分析。結果證實若將大量表現OsMAPK3之水稻經乾旱、高鹽、低溫和巴拉刈處理後，除葉綠素含量、Fv/Fm比值與存活率增加外，亦可提升脯胺酸含量與抗氧化酵素活性增加對逆境之耐受性。相反的，抑制表現OsMAPK3 水稻經相同處理後對逆境則不具耐受性。另外，大量表現OsMAPK3水稻在幼苗期經低溫處理後可促進分蘗，顯示轉殖水稻受低溫傷害時，可藉由增加分蘗數目以提高存活率，而轉殖水稻生長至成熟期時則可發現高節位分蘗現象，此高節位分蘗芽以插枝方式種植仍可正常生長抽穗，或許與老化下之延長生長有關。

本論文成功地將位於訊息傳遞上游的調控基因轉入於水稻中，並能有效地提升水稻對於不同逆境及除草劑之耐受性，相對於僅轉入訊息傳遞下游標的基因的作物，此OsMAPK3轉殖水稻不僅能提升作物對不同逆境之耐受性，亦可提升作物對除草劑之耐性而有利於田間管理。未來研究可針對轉殖株藉由生物晶片或2-D gel 方法，分析轉殖與非轉殖水稻間有那些基因或蛋白質，因大量或抑制OsMAPK3基因之表現而受影響，以此進一步分析OsMAPK3 基因在逆境下的調控模式，此外亦可探討OsMAPK3 對水稻分蘗相關基因之影響，乙烯及生長激素含量之變化，以明瞭OsMAPK3如何改變水稻之生長發育。總而言之，本論文以轉殖方式證實OsMAPK3基因參與水稻非生物逆境及除草劑耐受性之調控，並與水稻分蘗之形成有關。

關鍵字

非生物性逆境 ； 除草劑 ； 水稻 ； 過度表現 ； RNA干擾 ； 葉綠素螢光 ； 脯胺酸 ； 抗氧化酵素 ； 分蘗（分枝）

並列摘要

Mitogen-activated protein kinase (MAPK) is one of the important components that know to participate in regulation of plant abiotic-and boitic- stress responses and in controlling of plant growth and development. MAPK genes belong to a multiple gene family. However, the physiological function of each individual MAPK in rice and how MAPK engages in various abiotic stresses tolerance have not yet completely determined. In this study, first we addressed the putative function of OsMAPK3 by making alignment of the amino acid and DNA sequences and conducted the phylogenetic relationship analysis of OsMAPK3 with other rice MAPK gene. Then to understand gene expression pattern of OsMAPK3, the OsMAPK3 promoter sequence was further analysis for identification of different cis-acting DNA elements. The result showed that OsMAPK3 and OsMAPK4 that already demonstrated to involve in plant abiotic stress tolerance are in the same group C. Their nucleotide sequences shared 69% similarity; while OsMAPK3 only share 19% to 51% of sequence similarities with other OsMAPK genes. Besides, several stress-, hormone- responsive and development-control related cis-acting DNA elements exist in the promoter of OsMAPK3, including DRE, HRE, CRE, ABRE, ARE, and Myb factor recognition site. When TNG67 rice seedlings were treated with different abiotic stresses and OsMAPK3 gene expression was determined, OsMAPK3 can be induced by ABA, NaCl, drought, H2O2 and SA. On the other hand, there is no effect of paraquat, CuSO4, CdCl2 and methyl jasmonate treatments
To reveal the effect of OsMAPK3 on abiotic stress tolerance of rice, we took Agrobacterium-mediated gene transfer method to produce both over-expressing and knockout OsMAPK3 transgenic rice plants and its effect on the tolerance capability of rice seedlings under drought and salt stresses was examined. The result indicated that over-expression of OsMAPK3 in rice lead to increase of higher survival ratio, chlorophyll content, Fv/Fm value, proline content and antioxidant activity under stress. This result provided conceivable functional evidence that over- expression of OsMAPK3 gene could improve the abiotic stress and herbicide tolerance. On the contrast, the OsMAPK3 knockout transgenic rice displayed intolerant phenotype against stresses. Moreover, the over-expression OsMAPK3 transgenic rice can promote new tiller formation under cold treatment and ends with new tiller at higher internodes position when reaching maturity. We concluded that these phenomena of new tiller formation may be related to the escape of cold damage and enhance survival of rice seedlings or delay the senescence to death of rice. In this thesis, we successfully transduced OsMAPK3 gene into rice and efficiently rise up the abiotic stress and herbicide tolerance. It will be worthy to note that compared to improve plant stress tolerance through transferring the signal transduction downstream gene, our approach seems better and benefit field management than traditional study.
In future, we hope to apply microarray and 2-D gel analysis techniques in characterization the gene and protein expression profiles between wild type and OsMAPK3 transgenic rice plants to understand the regulation mechanism of OsMAPK3 gene under stresses. In addition, we are dedicated to elucidate the effect of OsMAPK3 on branching (tiller)-formation related genes in rice as well as change the endogenous content of ethylene and auxin that lead to alteration of rice growth and development. Taken together, by transgenic approach this study demonstrated that OsMAPK3 gene can play an important role in regulation of abiotic stress and herbicide tolerance, also affect the tiller formation in rice.

並列關鍵字

abiotic stresses ； herbicide ； rice (Orzya sativa L.) ； overexpression ； RNA interference ； chlorophyll fluorescence ； proline ； antioxidative enzyme ； tilling (branching)

參考文獻

趙雲洋、 劉麗飛、張孟基 (2007a) 抑制OsMAPK3基因表現會降低水稻對高鹽與乾旱的耐受性。台灣農業化學與食品科學。 45（3）：133-145。

趙雲洋、 劉麗飛、張孟基 (2007b) 離層酸誘導之OsMAPK3水稻轉殖株可提昇巴拉刈之抗性。作物、環境與生物資訊。 4：185-200。

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Agrawal, G.K., Rakwal, R. and Iwahashi, H. (2002) Isolation of novel rice (Oryza sativa L.) multiple stress responsive MAP kinase gene, OsMSRMK2, whose mRNA accumulates rapidly in response to environmental cues. Biochem. Biophys. Res. Commun. 294: 1009-1016.