臺灣博碩士論文加值系統

矮南瓜黃化嵌紋病毒(Zucchini yellow mosaic virus, ZYMV)和木瓜輪點病毒西瓜型(Papaya ringspot virus type watermelon, PRSV-W)，屬馬鈴薯Y群病毒(genus Potyvirus)，為台灣地區洋香瓜（Cucumis melo L.）栽培期間最嚴重病毒病害之ㄧ，經常是限制栽培的一個重要因素。本試驗目的為運用農桿菌基因轉殖法，將ZYMV和PRSV-W的鞘蛋白基因轉殖至洋香瓜，以達到防治該病毒之目的。利用聚合酶連鎖反應和限制酶酵素切接，將ZYMV-7和PRSV-W鞘蛋白基因分別或共同構築於兩個轉殖載體pCAMBIA 1304和pCAMBIA 2301，再利用電穿孔方式將質體送入農桿菌Agrobactrium tumefaciens EHA 105。洋香瓜“秋香”品系的子葉培植體於含有1 mg/L BA和30 μM AgNO3的MS培養基再生率可達70%。以具有轉植載體的農桿菌感染子葉後共培養四天，培植體皆有GUS暫時性表現。培植體培養於篩選再生培養基上有18-39 %不定芽團產生。經PCR檢測結果證實，6個轉殖系具有ZYMV和PRSV-W雙鞘蛋白基因和2個轉殖系具有ZYMV-7鞘蛋白基因的轉殖植株，另經RT-PCR和ELISA測試轉基因的表現，證實無法偵測到轉錄之RNA和轉譯之蛋白質，後續將進行R0代接種ZYMV和PRSV-W抗病毒評估。

Abstract

Melon (Cucumis melo L.) is an important crop of Cucurbitaceae. It is susceptible to both Zucchini yellow mosaic virus (ZYMV) and Papaya ringspot virus type watermelon (PRSV-W) which are limiting factors of the production of melon. The application of biotechnological tools in melon breeding have attracted increasing interest and provided a valuable complementary conventional improvement of this species. In constructs of pCAMBIA 1304 or pCAMBIA 2301 binary vector containing ZYMV, PRSV-W or both coat protein gene constructs were mobilized from Escherichia coli into the disarmed Agrobactrium tumefaciens strain EHA 105 by eletroporation. In GUS test, cotyledon via Agrobactrium-mediated gene transformation showed strong GUS transient expression as visualized 4 day after co-cultivation. In melon transformation experiment result, explant regeneration only appeared in kanamycin selection medium but not in hygromycin selection medium. The putative transforants were confirmed by PCR using specific primers to CP gene, 35S promoter and NOS terminator. PCR positive plants of two lines that containing ZMMV-7 CP gene and six lines that containing both CP gene were selected. RT-PCR and ELISA analysis for those transgenic melons showed negative reaction. Virus resistance of the transgenic melons were testing.

中文摘要........................................................I
英文摘要.......................................................II
壹、前言........................................................1
貳、材料與方法..................................................12
一、病毒的來源、病毒核酸之萃取與實驗菌株及質體......................12
(一)病毒的來源..................................................12
(二)病毒核酸之萃取..............................................12
(三)病毒核酸電泳膠體分析.......................................13
(四)實驗菌株及質體............................................13
二、ZYMV-7與PRSV-W鞘蛋白基因構築於pCAMBIA 1304和
pCAMBIA 2301植物轉殖系.......................................13

(一) ZYMV-7與PRSV-W鞘蛋白基因選殖.............................14
(二) ZYMV-7與PRSV-W鞘蛋白基因構築於pGEM-T質體..................15
(三) ZYMV-7與PRSV-W單鞘蛋白基因構築pCAMBIA 1304質體............17
(四) ZYMV-7鞘蛋白基因單獨構築於pCAMBIA 2301質體................20
(五) PRSV-W鞘蛋白基因片段構築於pCAMBIA 2301質體................22
(六) ZYMV-7與PRSV-W鞘蛋白基因片段共同構築
pCAMBIA 1304和pCAMBIA 2301質體…..........................23
(七) pCAMBIA 1304-ZY、-PR、-PZ和pCAMBIA 2301-ZY、-SPRN、
-PZ質體轉型至農桿菌菌株EHA 105...............................25
三、農桿菌轉殖洋香瓜系統的建立...................................27
(一)洋香瓜不定芽再生系統的建立..................................27
(二)洋香瓜篩選抗生素濃度選定....................................28
(三)農桿菌媒介基因轉殖洋香瓜....................................28
四、轉殖植株檢測................................................29
(一) GUS檢測法................................................30
(二) PCR檢測ZYMV-7鞘蛋白基因、PRSV-W鞘蛋白基因、
35S啟動子和NOS終結子基因片段...................................30
(三)RT-PCR檢測擬轉殖ZYMV-7、PRSV-W鞘蛋白基因.....................32
(四)轉殖植株之酵素結合抗體檢定法分析..............................33
參、結果.........................................................35
肆、討論.........................................................61
伍、參考文獻......................................................67
附錄ㄧ…………………………………………………………………….. 76
附錄二…………………………………………………………………….. 77

九十三年我國農產貿易統計結果。
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