臺灣博碩士論文加值系統

蕓苔屬蔬菜在台灣有著重要的民生及經濟地位，但因夏季氣候高溫多濕，易造成病蟲害危害嚴重。為了防治蟲害，農民每年花費相當高的成本在田間噴灑農藥，但過度的施用農藥，不但對蟲產生了抗藥性，每年也需要花費更高的成本來做害蟲防治的工作，除此之外，農民的健康也會因可能吸入太多的農藥而造成損害。因此培育抗蟲害的蕓苔屬蔬菜品種，是改善台灣夏季蔬菜生產的重要研究工作。
蘇力菌(Bacillus thuningiensis, bt) 殺蟲晶體蛋白(insecticidal crystal proteins, ICP) 是開發甚早且效果與安全性廣受肯定的生物性農藥之一，目前利用植物基因工程，轉移bt基因，培育出抗蟲作物，已有許多報導，且部分已商品化生產。以植物葉綠體基因組 (plastid genome, plastome) 為基因轉殖目標，是目前植物基因轉殖的新趨勢。此外，將多個性狀或多個基因同時送入植物基因組的多基因轉殖，已成為作物育種的新趨勢。本研究係將以prrn為啟動子的cry1Ab及cry1Ac基因、cry1Ab及cry1C基因、cry1Ac及cry1C基因等bt基因，藉由基因槍法 (particle bombardment) 轉殖至結球白菜的葉綠體。本研究的目的為建立結球白菜之葉綠體基因共同轉殖系統，並探討利用葉綠體基因轉殖培育出抗蟲害及不會造成基因污染之結球白菜的可行性。
本研究將pCHL-1Ab-1Ac (cry1Ab及cry1A基因)、pCHL-1Ab-1C (cry1Ab及cry1C基因)及pCHL-1Ac-1C (cry1Ac及cry1C基因)等質體藉由基因槍法轟擊至''玉冠''結球白菜葉片的葉綠體，篩選對 10 ppm Spectinomycin 有抗性的再生轉殖植株。轉殖植株均經由PCR、南方墨點雜交、RT-PCR及西方墨點雜交等分析進一步確認。試驗結果顯示有二株轉殖植株為同時能表現cry1Ab 與cry1C 基因 (CCBT-02) 及cry1Ab 與cry1Ac 基因(CCBT-05)的共同bt轉殖結球白菜，殺小菜蛾幼蟲效果可達到100%；有一株轉殖植株為cry1Ab 與cry1Ac 基因(CCBT-05)共同轉殖的結球白菜，但僅能表現cry1Ab基因，也具有殺蟲效果；另一株轉殖植株(CCBT-03)為cry1Ab 與cry1C 基因共同轉殖的結球白菜，但均無法偵測到其各別基因之表現，殺蟲效果則達60%；而不是基因轉殖植株(CCBT-04)及未轉殖對照組植株 (CK)則受到小菜蛾幼蟲啃食，危害情形十分嚴重。

Brassica vegetables play an important role in livelihood of the people and the economic status in Taiwan. In summer, the high temperature and humidity cause very serious and plant diseases in combination with pesis and insects. In order to prevent and control the and insect pest, the farmers spend quite high cost to spray the agricultural chemicals every year in the field.But excessively use of pesticides, not only rsults in resisternce to cheimicals in the insect, also makes a higher cost every year to prevent and conteol the insect. On the other hand, the excessive use of pesticides has caused significant human health problems and environmental damage. Therefore, development of more insect­resistant Brassica varieties is considered to be of great economic importance in Taiwan.
Bacillus thuningiensis (Bt) produces a variety of insecticidal crystal proteins (ICPs) upon sporulation which are highly toxic to several insects. Different ICP genes of Bt have been successfully engineered into many crop-plants to obtain resistance against Lepidopteran insects. Chloroplast transformation technologies are a promising tool in biotechnology. On the basis of the increase in activities of multiple Bt proteins providing plants with better insect-defense capability, we propose to transfer cry1Ab, cry1Ac and cry1C genes into the chloroplast of Chinese cabbage. The objectives of this study are to establish the chloroplast gene transfer technology of Chinese cabbage and to study the possibility for improvement of Brassica vegetables with insect resistance and reduce the gene pollution via chloroplast transformation.
The cry1Ab+cry1Ac (pCHL-1Ab-1Ac), cry1Ab+cry1C (pCHL-1Ab-1C), or cry1Ac+cry1C (pCHL-1Ac-1C) genes were transferred into the ''Jade Crown'' cabbage chloroplast via particle gun mediated transformation. Transgenic plants were confirmed by resistance to 10 ppm of spectinomycin. The results of PCR, Southern hybridization, RT-PCR and Western hybridization analysis indicated that the transformed bt genes were integrated into the chloroplast genome and expressed its mRNA and protein. High degree of resistance to the Plutella xylostella were found in the transformed plants containing either one (CCBT-05, expressed cry1Ab) or two bt genes (CCBT-02: expressed cry1Ab and cry1C ; CCBT-05: expressed cry1Ab and cry1Ac).

總 目 錄
中文摘要..................................................i
英文摘要.................................................ii
目錄....................................................iii
圖表目次.................................................iv
前言......................................................1
前人研究..................................................3
ㄧ、葉綠體基因轉殖之研究..................................3
二、蘇力菌殺蟲晶體蛋白之研究..............................7
三、蕓苔屬蔬菜之基因轉殖之研究...........................12
材料方法.................................................15
結果.....................................................26
ㄧ、葉綠體bt基因轉殖載體之檢驗...........................26
二、Bt蛋白表現載體(pET30a-Ab、pET-30a-Ac 與pET30a-1C)之構
築及其表現分析........................................26
三、以基因槍轟擊cry1Ab、cry1Ac及cry1C基因至結球白菜及葉片
誘導植株再生.........................................27
四、篩選及檢驗轉殖cry1Ab、cry1Ac及cry1C 基因之結球白菜...28
討論.....................................................63
一、Bt蛋白表現載體(pET30a-Ab、pET-30a-Ac 與pET30a-1C)之構
築及其表現分析.......................................63
二、以基因槍轟擊cry1Ab、cry1Ac及cry1C基因至結球白菜葉片誘
導植株再生...........................................63
三、篩選及檢驗轉殖cry1Ab、cry1Ac及cry1C 基因之結球白菜...65
四、轉殖結球白菜植株抗蟲特性分析.........................67
參考文獻.................................................69

圖表目次
表一、不同年齡與部位之培殖體對''玉冠''結球白菜癒傷組織誘導
及芽梢再生的影響...................................32
表二、轉殖bt基因(cry1Ab、cry1Ac及cry1C)的''玉冠''結球白菜經
PCR、南方墨點雜交、RT-PCR及西方墨點雜交分析之結果..33
表三、小菜蛾餵食轉殖Bt基因(cry1Ab、cry1Ac及cry1C)的''玉冠
''結球白菜再生植株葉片，72小時後，死亡的百分比.......34
圖一、以限制酵素 pstⅠ及kpnⅠ切割攜帶 bt 基因之甘藍葉綠體
轉殖載體(pCHL-1Ac-1Ab、pCHL-1Ac-1C、pCHL-1Ac-1C) 之
情形...............................................35
圖二、以PCR檢測pCHL-1Ac-1Ab、pCHL-1Ac-1C及 pCHL-1Ac-1C載
體之正確性.........................................36
圖三、pET30a-1Ab之構築流程及限制酵素圖譜.................37
圖四、pET30a-1Ac之構築流程及限制酵素圖譜.................38
圖五、pET30a-1C之構築流程及限制酵素圖譜..................39
圖六、pET30a-1Ac、pET30a-1Ab及pET30a-1C等蛋白表現載體以限
制酵素切割後，經電泳分離出不同大小片段.............40
圖七、以PCR檢測pET30a-1Ac、pET30a-1Ab及pET30a-1C之蛋白表
現載體正確性.......................................41
圖八、以SDS-PAGE分析cry-1Ab基因在E. coli BL-21(DE3)中表現
的情形.............................................42
圖九、以SDS-PAGE分析cry-1Ac基因在E.coli BL-21(DE3)中表現
的情形.............................................43
圖十、以SDS-PAGE分析cry-1C基因在E.coli BL-21(DE3)中表現的
情形...............................................44
圖十一、以SDS-PAGE分cry-1Ab、cry-1Ac及cry-1C基因在E.coli
BL-21(DE3)中表現的情形(A), 及以化學冷光西方墨點雜交
分析cry1Ab、cry1Ac及cry1C蛋白質的表現的情形(B).....45
圖十二、9天齡子葉(A)、14天齡葉片(B)、27天齡葉片(C)之''玉
冠''結球白菜培殖體的再生情形，及14天齡葉片之芽梢再
生情形(D) ........................................46
圖十三、''玉冠'' 結球白菜之再生芽體培養在含有不同濃度的
spectinomycin的再生培養基，生長21 天之前(上排)及
之後(下排)的情形。Spectinomycin 的濃度分別為0(A)、
5(B)、10(C)及15ppm(D)............................47
圖十四、結球白菜葉綠體基因轉殖再生情形...................48
圖十五、轉移bt基因(pCHL-1Ac-1Ab、pCHL-1C-1Ac 及 pCHL-1Ab-
1C )的''玉冠''結球白菜，以引子 A 1、A2 分析aadA 基
因，經PCR反應之產物在電泳膠片上分離之情形。CK：未
轉殖''玉冠''結球白菜...............................49
圖十六、轉移bt基因(pCHL-1Ac-1Ab及 pCHL-1Ab-1C)的''玉冠''結
球白菜，以引子3、4分析cry1Ab 基因，經PCR反應之產
物在電泳膠片上分離之情形。CK：未轉殖''玉冠''結球白
菜...............................................50
圖十七、轉移bt基因 (pCHL-1Ac-1Ab 及 pCHL-1C-1Ac) 的’玉冠’
結球白菜，以引子1、2分析cry1Ac 基因，經PCR反應之
產物在電泳膠片上分離之情形。CK：未轉殖’玉冠’結
球白菜...........................................51
圖十八、轉移bt基因 (pCHL-1C-1Ac 及 pCHL-1Ab-1C ) 的''玉冠''
結球白菜，以引子 5、6 分析cry1C 基因，經PCR反應之
產物在電泳膠片上分離之情形。CK：未轉殖''玉冠''結球
白菜.............................................52
圖十九、轉移 aadA 基因的’玉冠’結球白菜，其DNA以限制酵素
Pst I及KpnⅠ切割，再以aadA的PCR產物作為探針經南方
墨點雜交法分析之情形。CK：對照組.................53
圖二十、轉移 cry1Ab基因的''玉冠''結球白菜，其DNA以限制酵素
Pst I切割，再以cry1Ab 的PCR產物作為探針經南方墨點
雜交法分析之情形。CK：對照組.....................54
圖二十一、轉移 cry1Ac 基因的’玉冠’結球白菜，其DNA以限制
酵素KpnI切割，再以cry1Ac 的PCR產物作為探針經南
方墨點雜交法分析之情形。CK：對照組.............55
圖二十二、轉移 cry1C基因的’玉冠’結球白菜，其DNA以限制酵
素PstI切割，再以cry1C 的PCR產物作為探針經南方墨
點雜交法分析之情形。CK：對照組..................56
圖二十三、轉移bt基因 (pCHL-1Ac-1Ab 、pCHL-1C-1Ac 及 pCHL-
1Ab-1C)的’玉冠’結球白菜，以引子 A 1、A2 分析
aadA mRNA，經RT-PCR反應之產物在電泳膠片上分離之
情形。CK：未轉殖’玉冠’結球白菜................57
圖二十四、轉移bt基因 (pCHL-1Ac-1Ab及 pCHL-1Ab-1C) 的''玉
冠''結球白菜，以引子3、4分析cry1Ab mRNA，經RT-PCR
反應之產物在電泳膠片上分離之情形。CK：未轉殖''玉
冠''結球白菜.....................................58
圖二十五、轉移bt基因(pCHL-1Ac-1Ab 及 pCHL-1C-1Ac ) 的''玉
冠''結球白菜，以引子1、2 分析cry1Ac mRNA，經RT-
PCR反應之產物在電泳膠片上分離之情形。CK：未轉殖
''玉冠''結球白菜...................................59
圖二十六、轉移bt基因 (pCHL-1C-1Ac 及 pCHL-1Ab-1C ) 的''玉
冠''結球白菜，以引子 5、6 分析cry1C mRNA，經RT-PCR
反應之產物在電泳膠片上分離之情形。CK：未轉殖''玉
冠''結球白菜.....................................60
圖二十七、轉殖 bt基因(pCHL-1Ac-1Ab、pCHL-1Ab-1C、pCHL-1C-
1Ac)的''玉冠''結球白菜，以化學冷光西方墨點雜交分析
cry1Ab (A)、cry1Ac (B) 及cry1C (C) 蛋白質表現的
情形。萃取轉殖bt基因的結球白菜再生殖株之蛋白質經
SDS-PAGE 分離，並以蘇力菌晶體蛋白多株抗體偵測。
CK：未轉殖植株..................................61
圖二十八、轉殖cry1Ab、cry1Ac 及cry1C 基因 (pCHL-1Ab-1C、
pCHL-1Ab-1Ac及pCHL-1C-1Ac) 的''玉冠''結球白菜，及
對照組(CK)的葉片，餵食菜蛾72小時前(上)及後(下)，
小菜蛾危害葉片之情形。分別餵食：CK 8隻；CCBT-01 4
隻；CCBT-02 3隻；CCBT-03 6隻；CCBT-04 9隻；CCBT-
05 5隻小菜蛾.....................................62

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