臺灣博碩士論文加值系統

分離自以離素(abscisic acid, ABA)處理的大麥（Hordeum vulgare L.）粉糊層(aleurone layer)的HVA1蛋白，屬於第三類的LEA(Late Embryogenesis Abundant)蛋白質，大小約為22kDa。研究發現，自土壤吸水三天的植物幼苗，在離素、乾旱、冷害、鹽害及高溫等逆境處裡下，能夠迅速的表現出HVA1蛋白質。水稻為最早利用HVA1轉殖的植物，HVA1基因轉殖水稻發現較能適應高鹽的逆境。到目前為止，已有許多HVA1基因轉殖作物被發表，例如：菸草(tobacco)、蕃茄(tomato)、小麥(wheat)及燕麥(oat)等，多為糧食作物或模式，尚無HVA1轉殖花卉的問世。因此本實驗利用農桿菌轉殖菊花，得到HVA1基因轉殖菊花，並比較轉殖株與栽培種菊花在鹽逆境下之抗氧化基因表現與生理型態之差異。經農桿菌轉殖後，獲得多株再生成功的菊花幼苗，選取具有抗生素抗性之擬轉殖菊花，經過PCR檢測確認獲得數個轉殖株。將切離葉片以鹽逆境處理，經RT real-time PCR結果顯示，以轉殖株400mM NaCl處理十分鐘後，catalase基因即被誘導表現。而且catalase基因不但有週期性表現之現象，而且表現量逐漸增加。比較鹽逆境處理前後catalase基因的表現量，發現栽培種菊花之catalase基因表現量下降98%，而轉殖株之catalase基因表現量，最低下降92%，最高則上升至214%。在catalase活性表現方面，轉殖株的catalase活性增加量均比栽培種高，栽培種之catalase活性增加量僅0.29，轉殖株的catalase活性增加量在1.63至4.31之間。由此得知，我們所獲得之HVA1基因轉殖菊花在鹽逆境下，其catalase基因的表現程度較野生株強，具較佳之抗氧化活性。

The barley HVA1 is belong to group 3 LEA (late embryogenesis abundant ) protein and its molecular weight is about 22kDa. The HVA1 protein was firstly isolated from the aleurone layer of the barley seed. HVA1 mRNA was rapidly induced in young seedlings (3 days after imbibition) by ABA treatment and a series of stress conditions, such as drought, cold, NaCl, and heat. The HVA1 transgenic rice harboring HVA1 gene was found to be more tolerant to the salt stress than wild-type. In order to increase the salt tolerance of chrysanthemum, we try to produce transgenic chrysanthemum by Agrobacterium-mediated transformation. After Agrobacterium transformation, we have obtained a lot of chrysanthemum seedlings. By putting detached leaf of chrysanthemum seedling in a selection medium, we acquired several chrysanthemum plants which may contain HVA1 gene. These putative transgenic plants were confirmed by PCR analysis. By the analysis of RT real-time PCR, the expression of catalase gene of transgenic plant was found to induce when the detached leaf put in salt stress for 10 min. Morever, we find that catalase gene is regulated by a circadian rhythm, and the expression of catalase increases with every rhythm cycle. Compared with the extent of catalase expression before and after salt stress treatment, the catalase of wild-type reduces 98%, while transgenic plants are from -92% to 214%. Detecting the catalase activity of detached leaf treated with salt stress or water, we find that the increase of catalase activity in transgenic plant is higher than that in wild-type plant. The catalase cativity only increase 0.29 in wild-type, however, the catalase cativity increase from 1.63 to 4.31 in transgenic plants. We conclude that the transgenic chrysanthemum plants containing HVA1 have higher antioxidant ability under salt stress.

目錄
中文摘要.....................................................i
英文摘要.....................................................ii
目錄.........................................................I
圖目錄.......................................................III
前言.........................................................1
鹽害與乾旱....................................................1
耐鹽與耐旱植物培育的相關研究....................................2
滲透質基因之轉殖應用...........................................2
抗氧化基因之轉殖應用...........................................3
離子轉運蛋白之基因轉殖在耐鹽育種上的應用..........................3
功能未知的抗旱基因之轉殖應用....................................4
LEA蛋白質....................................................4
LEA蛋白質之結構...............................................5
HVA1蛋白質...................................................6
含HVA1基因轉殖植物之相關研究....................................6
Real-time PCR................................................7
Real-time PCR在植物研究的應用..................................9
植物致病菌或共生菌之偵測........................................9
加工食品之外源DNA偵測......................................9
菊花簡介......................................................10
菊花之栽培特性.................................................10
菊花之新種培育.................................................11
菊花之農桿菌轉殖...............................................12
材料與方法....................................................13
含CaMV35S啟動子之表現載體，pMON530/HVA1.........................13
細菌質體抽取...................................................13
含植物表現載體pMON530/HVA1之農桿菌轉型..........................14
菊花癒傷組織之培養.............................................14
菊花組織的轉殖.................................................15
農桿菌之生化檢測-判定農桿菌之真偽................................15
菊花轉殖株之篩選...............................................15
促進菊花轉殖株根系的生長........................................15
菊花DNA之萃取.................................................16
以PCR檢測擬轉殖菊花............................................16
轉殖後菊花無菌苗之馴化與室外栽種.................................17
菊花RNA之萃取.................................................17
RNA之反轉錄(Reverse Transcription)反應........................18
Real-time PCR之進行...........................................18
南方氏墨點法...................................................19
過氧化氫酶(catalase)活性測試....................................21
菊花葉片之耐鹽測試..............................................22
實驗結果.......................................................23
菊花轉殖後之生長及篩選測試.......................................23
菊花轉殖株HVA1基因之鑑定........................................23
Real-time PCR之結果判別........................................24
鹽逆境處理下之catalase基因表現...................................25
鹽逆境處理下之catalase活性表現...................................25
鹽逆境處理後，切離葉片的回復情形..................................26
討論..........................................................27
HVA1轉殖菊花之判定.............................................27
菊花DNA與RNA之萃取..............................................27
Catalase基因在RNA與蛋白質層次的表現..............................28
參考文獻.......................................................31
附錄...........................................................55
作者簡歷.......................................................62

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