臺灣博碩士論文加值系統

　　釩及鋅是動物及植物生長所需之必要元素，然而在水稻中關於釩及鋅所造成的逆境訊息傳遞之研究卻甚為匱乏。過去研究發現，在植物組織中若累積過量的重金屬，常會導致活性氧化物質 (reactive oxygen species, ROS) 大量產生，而重金屬產生的逆境訊息，常會利用MAPK (mitogen-activated protein kinase) 作為訊息傳遞物質。本研究主要探討蛋白質酪胺酸去磷酸酶 (protein tyrosine phosphatase, PTP) 抑制劑－釩及鋅在水稻根部細胞訊息傳遞途徑中所扮演的角色。當以高濃度 (20 mM) 的釩處理水稻植株時，會造成明顯的根部細胞死亡現象以及抑制根的生長。而分別用釩及鋅處理水稻植株時，則會明顯的引發MBP (myelin basic protein) 激酶活性。進一步以蛋白質免疫沉澱實驗分析，證實受釩及鋅所誘發之分子量40與42 kDa的MBP激酶為MAPKs。若以sodium benzoate，一種ROS清除劑做前處理，結果發現釩所造成的細胞死亡會受到抑制，而釩與鋅所誘發的MAPKs活性也有被抑制的現象。另外，利用蛋白質去磷酸酶抑制劑－cantharidin及endothall處理，也會對於釩及鋅所誘發的MAPKs活性產生抑制的情況。綜合以上研究結果，我們推論ROS及蛋白質去磷酸酶皆參與釩及鋅所誘發的MAPKs訊息傳遞途徑。經由序列比對分析，我們在水稻中發現四個蛋白質酪胺酸去磷酸酶，它們皆帶有一段高度保留性的活化區。除此之外，在組織特異性分析中，OsPTP1會在根、葉及穗狀花序中表現，而且其表現量不會受到釩或鋅逆境影響。在本研究中亦利用pyronitrophenyl phosphatase (pNPP) 為受質，探討OsPTP1的酵素活性。

　　Vanadium and zinc have been reported to be essential nutrition elements in animals and plants, however, the vanadium and zinc stress signal transduction pathways in rice are poorly investigated. Previous studies reported that the excess accumulation of heavy metal often accompanies with the reactive oxygen species (ROS) overproduced, and the transduction of metal stress signals may through the MAPKs (mitogen-activated protein kinases). This study is set to clarify the role of protein tyrosine phosphatase (PTP) inhibitors, vanadium and zinc, on rice root signal transduction pathways. We show that higher concentration (20 mM) of vanadium reduced rice root cell viability and suppressed the root growth. Vanadium and zinc elicited a remarkable increase of MBP kinases activities. By immunoblot analysis, we suggest that 40 and 42 kDa MBP kinases activated by vanadium and zinc are MAPKs. The ROS scavenger, sodium benzoate, was able to effectively prevent vanadium caused cell death and reduced the activities of vanadium- and zinc-induced MAPKs. The protein phosphatase inhibitors, cantharidin and endothall, were also able to prevent vanadium- and zinc-induced MAPks activation. Thus, we suggest that ROS and protein phosphatase may function in the vanadium- and zinc-triggered MAPK signalling pathway in rice roots. With the BLAST (basic local alignment search tool) analysis, we identify four tyrosine-specific PTPs in rice genome. Each contains a predicted catalytic domain containing sequence motifs characteristic of tyrosine-specific PTPs. The tissue specific expression analysis shows that OsPTP1 was expressed in rice root, leaf, and panicle, and the transcription of OsPTP1 in rice root was not been affected by vanadium and zinc. The pyronitrophenyl phosphatase (pNPP) was used as substrate and the phosphatase activity of OsPTP1 was measured in the present study.

中文摘要 i
Abstract ii
Abbreviations iii

Introduction 1
The effect of vanadium in eukaryotes 1
Cytotoxicity of vanadium on plants 2
Cytotoxicity of zinc on plants 3
Vanadium and zinc induce reactive oxygen species (ROS) generation in eukaryotes 4
MAPK signal transduction pathways in eukaryotes 5
MAPKs involve in vanadium and zinc stress signal transduction pathways 5
Protein phosphatases in eukaryotes 6
Protein tyrosine phosphatases in eukaryotes 7
Protein tyrosine phosphatases in plants 8
The effect of vanadium and zinc on PTP activites 9
Aims of this study 9

Materials and Methods 10
Plant materials 10
Evaluation of cell death using Evans blue assay 10
Root length determination 11
Reactive oxygen species generation determination 11
Preparation of homogenates 11
Immunoblot analysis 12
In gel kinase activity assay 13
Immunoprecipitation assay 13
Preparation of total RNA 14
Identification and expression plasmids construction 15
Over expression of the GST fusion protein in E.coli 15
GST-fusion protein purification 16
Protein tyrosine phosphatase activity assay 16
Satistical analyses 17

Results 18
The effect of vanadate on the root growth of rice seedling 18
The effect of zinc on the root growth of rice seedling 18
The effect of vanadate on rice root cell viability 18
The induction of MAPKs activities by vanadate and zinc 19
The reactive oxygen species (ROS) scavenger, sodium benzoate, reduced vanadate induced rice root cell death 20
The effect of protein phosphatases on vanadate- and zinc-induced MAPKs
activities 20
Molecular cloning and identification of the Oryza sativa protein tyrosine
phosphatase 21
Expression of the OsPTP1 gene 22
The OsPTP1 gene encodes a non-function tyrosine-specific PTP 22

Discussion 24
References 29

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