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標題: 刺角瓜抗木瓜輪點病毒候選基因Cm8及Cm23選殖與分析
Molecular cloning and analysis of papaya ringspot virus resistance candidate genes Cm8 and Cm23 in Cucumis metuliferus
作者: Szu-Yun Chen
關鍵字: Cucumis metuliferus;papaya ringspot virus (PRSV);Nicotiana benthamiana;Solanum lycopersium;heat shock protein 40;casein kinase I;刺角瓜;木瓜輪點病毒;圓葉菸草;番茄;heat shock protein 40;casein kinase I
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刺角瓜(Cucumis metuliferus)為原產於西非的水果,對許多瓜類病蟲害有極佳抗性或耐性,是良好的抗病基因來源,刺角瓜品系PI292190對於會嚴重影響作物產量的木瓜輪點病毒(papaya ringspot virus, PRSV)具有抗性,本論文自此品系中篩選出可能的參與刺角瓜抗病反應的候選基因Cm8及Cm23基因片段,並利用干擾性核醣核酸(RNA interference, RNAi)技術,將帶有正反向Cm8或Cm23基因片段的載體,轉殖入PI292190中,以逆向遺傳學的原理分析候選基因之功能。此外,分析Cm8及Cm23基因在刺角瓜接種PRSV後的表現,發現Cm8及Cm23基因接種PRSV後表現量都有上升的情形。本論文也以genome walking及快速增幅cDNA末端快速擴增cDNA末端(rapid-amplification of cDNA ends, RACE)等技術,選殖Cm8及Cm23基因的全長片段,並於資料庫中進行序列比對分析,以及序列編碼的蛋白功能預測,結果發現Cm8基因為編碼heat shock protein 40 (HSP40)類型的蛋白,Cm23基因則為編碼casein kinase I (CKI)類型的蛋白。進一步將刺角瓜的Cm8及Cm23基因全長的片段構築於過表現載體中,並轉殖入圓葉菸草(Nicotiana banthamiana)及番茄(Solanum lycopersium)中,未來,在獲得轉殖株後,將繼續進行後代分析及病毒接種,期望以功能性互補試驗證明候選基因與異源植物抗病毒功能的相關性。

Horned melon (Cucumis metuliferus) is endemic in southern and central Africa, where it is eaten as fruit line, PI 292190 (L37), shows strong resistance to papaya ringspot virus (PRSV) causing serve damages to the yield and fruit quality of papaya and cucurbits. In this study, Cm8 and Cm23 genes, potentially involving PRSV-resistance, were isolated from L37. To investigate their resistant function against PRSV, RNA interference (RNAi) containing sense and antisense Cm8 or Cm23 gene fragments, were transformed into L37. In addition, Cm8 or Cm23 gene were all induced in C. metuliferus after inoculating by PRSV. Rapid-amplification of cDNA ends (RACE) and Genome walking were conducted to obtain full-length Cm8 and Cm23 cDNA and genomic fragments. Cm8 gene was predicted to encode heat shock protein 40 (HSP40) protein, and Cm23 gene was predicted to encode casein kinase I (CKI) protein. To verify their relationships with disease resistance, complementation experiments were performed in which full-length cDNA and genomic fragments of both candidates were overexpressed in Nicotiana bethaminana and Solanum lycopersium. Transgenic plants will be generated and their PRSV-resistance will be verified in the future.
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