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Cloning and functional analysis of serine proteinase inhibitor of Cucumis metuliferus
|關鍵字:||Cucumis metuliferus;serine proteinase inhibitor;Papaya ringspot virus;biotic;abiotic;刺角瓜;絲氨酸蛋白酶抑制子;木瓜輪點病毒;生物逆境;非生物逆境||引用:||Belenghi, B., F. Acconcia, M. Trovato, M. Perazzoli, A. Bocedi, F. Poticelli, P. Ascenzi, and M. Delledonne. 2003. Atcyssi, a cystatin from Arabidopsis thaliana, suppresses hypersensitive cell death. Eur. J. Biochem. 270: 2593-2604. Birk, Y. 1996. Protein proteinase inhibitors in legume seeds-overview. Arch. Latinoam. Nutr. 44: 26-30. Chen, P., G. R. Buss, and S. A. Tolin. 1993. Resistance to soybean mosaic virus conferred by two independent dominant genes in PI 486355. J. Hered. 84: 25-28. Christeller, J. T. 2005. Evolutionary mechanisms acting on proteinase inhibitor variability. FEBS J. 272: 5710-5722. Connors, B. J., N. P. Laun, C. A. Maynard, and W. A. Powell. 2002. Molecular characterization of a gene encoding a cystatin expressed in the stems of American chestnut (Castanea dentata). Planta 215: 510-514. Davletova, S., L. Rizhsky, H. Liang, Z. 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蛋白酶抑制子廣泛存在於生物體中，可降低或抑制蛋白酶的活性進而參與生理現象調控及抗逆境相關反應。本研究從刺角瓜 (Cucumis metuliferus) 中選殖出CmSPI基因可編碼出絲氨酸蛋白酶抑制子，屬於potato I type 蛋白酶家族。經序列分析發現CmSPI基因上游區域具有core abscisic acid response element、T/GBOXATPIN2 element以及wound response element，推測與逆境反應相關。為了瞭解CmSPI在植物中的功能性，因此在生物逆境方面，首先利用RNA interference (RNAi) 的方式靜默刺角瓜抗木瓜輪點病毒 (papaya ringspot virus, PRSV) 品系PI 292190之CmSPI基因表現。在CmSPI基因靜默刺角瓜品系H1和H2中發現，PRSV可突破植物的防禦機制並於接種病毒21天後開始產生病徵。此外，將刺角瓜CmSPI基因轉殖於圓葉菸草 (Nicotiana benthamiana) 中，得到的轉基因菸草品系F-21與F-51，對馬鈴薯病毒Y (potato virus Y, PVY ) 具有抗性。在非生物逆境方面發現，在鹽分逆境下，過表現CmSPI基因菸草品系Y1與Y2之種子發芽比率相較於未轉殖對照組可提高55.6-73.3%，而在Mannitol乾旱逆境處理下則可提高63-75%。此外在PEG乾旱逆境處理下，轉基因品系Y2之葉圓片總葉綠素含量相較於未轉殖對照組提高10%。綜言之，本研究證明CmSPI基因確實與刺角瓜抗PRSV之抗病反應相關，並可提供圓葉菸草對於PVY之抗性，同時CmSPI可增加圓葉菸草於鹽分及乾旱下的耐受性。本研究的結果將有助於瞭解刺角瓜CmSPI基因在生物及非生物逆境中的所扮演的腳色，在未來可進一步運用於作物抗病及環境逆境防治。
Proteinase inhibitors (PIs) is an ubiquitous protein involved in physiology reaction, biotic and abiotic stress defense responses via block the active center or allosteric interaction with specific proteinase. In this study, we cloned Cucumis metuliferus serine PI (CmSPI) gene, a potato I type PIs, from horned melon (C. metuliferus) papaya ringspot virus (PRSV) resistant line PI 292190. A core abscisic acid response element, a T/GBOXATPIN2 element, and a wound response element were identified on the promoter region of CmSPI indicating its role in plant stress responses. To identify CmSPI function, CmSPI RNA interference (RNAi) transgenic horned melon lines, H1 and H2 were generated, and showed broken down of the resistance after challenged with PRSV at 21 dpi. Transgenic tobacco (Nicotiana benthamiana) lines expressing a full length of CmSPI genomic fragment, F21 and F51, showed resistance to one of potyvirus, potato virus Y (PVY), but not Biden mottle virus (BiMoV) and Turnip mosaic virus (TuMV). In addition, CmSPI overexpression tobacco lines, Y1 and Y2, showed tolerance of salt and drought stresses. The germination rate of the two transgenic lines increased dramatically about 55.6% to 73.3% higher than wild type (WT) under NaCl salt stress and increase of 63% to 75% than WT under Mannitol drought stress. In addition, the chlorophyll content of leaf discs in transgenic lines, Y2, was 10% higher than WT under PEG drought stress. These results suggested that CmSPI can response to biotic and abiotic stresses. This study provided the evidence for a better understanding of CmSPI in interaction with pathogens and environment stresses. This can be applied to crop disease-resistance and stress-resistance breeding in the future.
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