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Generation of Coat-Protein Transgenic Cucurbits Resistant to Aphid-Borne Potyviruses
台灣瓜類栽培面積廣大且種類繁多, 其中以西瓜及甜瓜為大宗.此二作物受矮南瓜黃化病毒( Zucchini yellow mosaic virus, ZYMV )及木瓜輪點病毒西瓜系統( Papaya ringspot virus W type, PRSV-W )之危害, 每每造成嚴重損害, 由於缺乏抗病材料, 傳統方法對於此二病毒的防治迄無良方, 本計畫乃利用遺傳工程方法構築具ZYMV及PRSV-W病毒之鞘蛋白轉基因瓜類, 預期應可作為抵抗病毒傳播之一個極具價值的辦法.以構築有NPT Ⅱ及矮南瓜黃化嵌紋病毒( Zucchini yellow mosaic virus, ZYMV )及木瓜輪點病毒西瓜型( Papaya ringspot virus W type, PRSV-W )鞘蛋白基因的農桿菌為轉殖媒介對西瓜及甜瓜商用栽培種的種子進行轉殖, 試圖獲得抗ZYMV及PRSV-W病毒的轉型植物.第一年計畫已經嘗試的轉殖法有”頂芽( apex )轉殖法”、”子葉切割轉殖法”、”植體上頂芽( apex )感染轉殖法”及”莖段切傷轉殖法”.利用”頂芽( apex )轉殖法”及”子葉切割轉殖法”對六千多個甜瓜種子進行轉殖, 初步產生5個含有ZYMV或PRSV-W鞘蛋白基因的轉型甜瓜株系, 在kanamycin培養基中生長良好並能以PCR偵測到病毒鞘蛋白基因; 西瓜經轉殖三-四千個種子後, 目前有芽體正篩選中, 但尚無成功轉型的植物產生.並非所有進行轉殖處理的品種都能順利產生轉型植物, 西瓜的轉型遠比甜瓜困難許多, 各個品種對於應用農桿菌進行轉型的反應各不相同.本年度對已轉型成功的株系將進一步進行微體繁殖, 及溫室的抗病接種實驗以評估其抗病性狀.同時有許多培養及轉殖的細節尚待改進以提高轉殖的效率, 甜瓜及西瓜不同品系基因轉殖工作亦將繼續進行, 另外將加強銀輝甜瓜品系之轉殖, 以期獲得較多之轉基因品系.
Zucchini yellow mosaic virus ( ZYMV )and Type W strain of Papaya ringspot virus ( PRSV-W )are transmitted by aphids and cause serious economical losses to cucurbits cultivation in Taiwan.The major objective of this study to was directed construct coat protein transgenic melon ( Cucumis melo )and watermelon ( Citrullus lanatus )that are resistant to infection by ZYMV and PRSV-W.Several transformation protocols have been used to regenerate commercial melon and watermelon cultivars.In the first year study, these regeneration schemes involved facilitating gene transfer by Agrobacterium tumefaciens with the reporter gene neomycin phosphortransferaseⅡ( NPT Ⅱ )and the ZYMV CP or PRSV-W CP gene.Four different methods have been developed for transformation of melon and watermelon, including apex infection, cotyledon cutting, In vivo apex infection, and stem wounding infection.A total of 5transformed R0lines were produced from six to seven thousand melon seeds.These R0lines survived well in the kanamycin selection medium and the presence of the CP gene was confirmed by PCR analysis.Three to four thousand watermelon seeds were also infected with A.tumefaciens and some transgenic calluses were produced under selection, but the transformed R0line has not been obtained.Our results showed that the transformation efficiency of melon and watermelon plants transformed by A.tumefaciens with ZYMV CP or PRSV-W CP gene varies from cultivars to cultivars.The regeneration process was found to be easier for melon than for watermelon.In this fiscal year, the 5transformated R0lines of melon will be micropropagated and challenged with either ZYMV or PRSV-W to evaluate their resistance to virus infection under greenhouse conditions.The transformation efficiency still has to be improved for melon and watermelon cultivars.More transgenic lines will be generated for the melon variety ”Silver Light”for greenhouse evaluation.
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