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研究生:林湛
研究生(外文):Chan Lin
論文名稱:綠豆抗蟲品系VC6089A及其抗蟲蛋白VrD1對四紋豆象之抗性作用與機制
論文名稱(外文):Characterization of the Resistance to Callosobruchus maculatus (F.) in a Mungbean Variety VC6089A and Its Resistance-Associated Protein VrD1
指導教授:洪淑彬洪淑彬引用關係
指導教授(外文):Shwu-Bin Horng
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:昆蟲學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:52
中文關鍵詞:VC6089A四紋豆象抗蟲蛋白VrD1
外文關鍵詞:Bruchid resistanceCallosobruchus maculatusVC6089AVrD1
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植物經過長期的演化,發展出一套防禦策略來抵抗病原微生物及植食性昆蟲的危害;但是隨著人類農業發展的介入,栽培作物逐漸失去其原本的防禦機制,造成疫病蟲害興起肆虐,使我們不得不想盡辦法來抗病除蟲。經由育種的技術,亞洲蔬菜研究與發展中心 (AVRDC) 培育出一種具有抗蟲活性的綠豆近等基因品系 (nearly isogenic line),VC6089A;並由中研院植物所陳慶三博士找到與抗性作用相關的抗蟲蛋白VrD1。本實驗室首先利用VC6089A綠豆種子飼養四紋豆象 (Callosobruchus maculatus (F.)),發現豆象族群死亡率高達96% 以上,這個結果顯示VC6089A抗蟲品系綠豆具有高度的抗豆象特性。不過將剩餘接近4% 的豆象存活個體連續飼養於抗蟲綠豆5代後,發現四紋豆象並沒有絕滅,並且可藉由高生殖力維持族群的正向增長。這個結果可能提供存活的豆象族群形成對此抗蟲綠豆產生抗性的機會,在未來VC6089A品系綠豆的應用上,仍須加強對豆象族群的監測與抗性管理。由人工種子添加4種濃度的VrD1進行生物檢測實驗中,發現隨著VrD1濃度提高,豆象的幼蟲發育期、成蟲體重以及雌蟲生殖力皆受到顯著的影響;而且VrD1濃度達0.2% (w/w) 以上就足以殺死所有的豆象幼蟲,這是目前所發現抗豆象作用最強的植物抗蟲蛋白之一。這個結果為未來VrD1應用於基因轉殖作物或開發為生物製劑提供重要的參考依據。另在抗性機制方面,實驗的結果顯示VrD1可能不是VC6089A抗蟲品系綠豆中主要的抗豆象物質,而其抗豆象機制初步推測可能兼具急性毒殺初齡幼蟲以及慢性抑制老熟幼蟲生長發育等角色,未來仍須進一步研究。
Characteristics of resistance of VC6089A, a mungbean (Vigna radiata) variety bred by utilizing a wild Vigna species, Vigna sublobata (Accession TC1966), and containing a novel protein VrD1, was investigated against cowpea weevil, Callosobruchus maculatus (F.). Seeds of VC6089A showed high level of resistance; more than 96% of the bruchid eggs failed to develop into adults, whereas over 80% of eggs laid on susceptible cultivar VC1973A became adults. Mortality of surviving bruchids raised for five generations on VC6089A remained higher than 96%; however, female adults maintained high fecundity through these generations. This positive growth of bruchid population reared on seeds of VC6089A may result in bruchid population eventually developing resistance to the resistant mungbean. The protein VrD1 purified from seeds of VC6089A showed marked toxicity to C. maculatus when insects were reared on artificial seeds containing varying levels of VrD1. Thorough inhibition of development was observed when artificial seeds containing merely 0.2% (w/w) VrD1 was used for insect feeding. VrD1 appears to be one of the most potent anti-bruchid proteins discovered from plant sources so far. But it seems not to be the principle factor responsible for bruchid resistance in VC6089A seed, considering the discrepancy between the toxicity of VrD1 and the resistant seed. The mechanism of VrD1 toxicity to C. maculatus needs to be investigated further. Our findings demonstrated the insecticidal activity of VC6089A seeds and VrD1 protein against C. maculatus. These results may facilitate safer control against bruchid infestation, but the potential of these pests developing resistance to the resistant mungbean variety through maintaining high fecundity should be carefully monitored and managed as well.
CHAPTER 1. Impact of VC6089A Resistant Mungbean Seeds on C. maculatus Populations……………………………………………………. 1
INTRODUCTION……………………………………………………..…. 1
MATERIALS AND METHODS……………………………………….… 3
Source of Mungbean Seeds and C. maculatus………………………… 3
Resistance of VC6089A Seeds to C. maculatus……………………….. 3
Survival and Reproduction of the Bruchids on VC6089A seeds for Five Consecutive Generations………………………………………. 4
Population Statistics for Three Generations of the Bruchid on VC6089A Seeds……………………………………………….….… 5
RESULTS………………………………………………………………… 6
Resistance of VC6089A Seeds……………………………………….... 6
Survival and Reproduction of the Bruchids on VC6089A for Five Generations……………………………………………………….…. 6
Population Statistics for Three Generations on VC6089A…………….. 7
DISCUSSION………………………………………………………….… 8
CHAPTER 2. Biological Effects of VrD1 Protein on C. maculatus………... 15
INTRODUCTION………………………………………………………... 15
MATERIALS AND METHODS…………………………………………. 17
Source of Protein and Bruchids………………………………………... 17
Bioassay of VrD1 with C. maculatus………………………………….. 17
RESULTS………………………………………………………………… 19
Effects of VrD1 on Survival and Development of C. maculatus……… 19
DISCUSSION……………………………………………………………. 21
CHAPTER 3. The Mechanism of Resistance in VC6089A………………… 31
INTRODUCTION………………………………………………………... 31
MATERIALS AND METHODS…………………………………………. 33
Source of Mungbean Seeds and Bruchids……………………………... 33
Investigation of C. maculatus-Resistant Activity in Decorticated or Artificial seeds of VC6089A………………………………………... 33
Histological Investigation of C. maculatus Larvae Feeding on VC6089A Seeds…………………………………………………….. 34
RESULTS………………………………………………………………… 35
Bruchid Resistant activity in Decorticated or Artificial seeds of VC6089A……………………………………………………………. 35
Histology of Bruchid Larvae Feeding on VC6089A Seeds…………… 35
DISCUSSION……………………………………………………………. 37
REFERENCES……………………………………………………………… 46
ACKNOWLEDGMENTS…………………………………………………... 52
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