Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/89165
標題: D型胺基酸氧化酵素(daao)、轉榖氨醯胺酵素(tga)及溶菌酶(lys)基因轉殖至小白菜之研究
Studies on Transformation of D-Amino Acid Oxidase (daao), Transglutaminase (tga), and Lysozyme (lys) Genes into Pak-choi (Brassica campestris L. ssp. chinensis (L.) Makino)
作者: Ming-Kun Chi
紀銘坤
關鍵字: D型胺基酸氧化酵素
轉榖氨醯胺酵素
溶菌酶
小白菜
d-amino acid oxidase
transglutaminase
lysozyme
Pak-choi
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摘要: 由於小白菜具有生長快速、高環境耐受性的優點,因此栽培容易,也較不受到氣候環境等自然條件的限制。小白菜具有重要的民生及經濟地位,是台灣可全年生產的重要葉菜類蔬菜之一。過去三十年來,雖然育種者在小白菜改良上卓有成效,但是受限於物種差異,傳統育種在少數幾種種原親本間能進行育種改良的部分已達極限,因此為了培育出更具有耐環境逆境與高品質的小白菜品種,提升小白菜的附加經濟價值與競爭力,利用基因轉殖技術培育新的小白菜品種是一很好的選項。 本論文第二章首先探討不同D型胺基酸的種類及濃度對甘藍、水稻、結球白菜、小白菜種子發芽與生長之影響。其目的為建立一個適當的D型胺基酸的種類及濃度,可有效的應用在daao篩選標誌基因,以建立一安全、完善的非抗生素藥劑(D型胺基酸)作為篩選轉殖植株的基因轉殖系統。本研究結果顯示20 mM的D-alanine、D-asparagine、D-methionine、D-lysine等4種不同D型胺基酸中,D-alanine及D-methionine強烈抑制甘藍、結球白菜與水稻的的發芽與生長,D-asparagine次之,D-lysine的抑制效果較少。完全抑制水稻、甘藍、結球白菜種子發芽與生長之4種D型胺基酸約為 20 mM D-alanine、40 mM D-methionine (甘藍80 mM D-methionine)、40~80 mM D-asparagine、125 mM D-lysine。D-alanine次致死濃度 (sub-lethal dose) 之研究顯示甘藍在5~10 mM、水稻在10~15 mM、小白菜在2.5~5 mM之間的D-alanine濃度有一明顯的抑制芽梢的生長,而後再提高D-alanine濃度即有嚴重抑制芽梢的生長與成活。本研究建議進行甘藍、水稻、小白菜轉殖培植體的初次(早期)篩選時,D-Ala濃度在次致死濃度之間,待轉殖培植體的組織狀態穩定後,再逐步提高D-Ala濃度,獲得再生轉殖植株的機率可大幅提昇。 本論文第三章探討利用daao基因作為轉殖植物篩選標誌基因之應用潛力。本研究之目的為:(一)、建立以 daao 基因作為甘藍、水稻與小白菜之農桿菌基因轉殖法的篩選標誌基因系統,(二)、建立以 daao 基因作為小白菜之基因槍基因轉殖法的篩選標誌基因系統。本研究已完成將篩選自三角酵母(Trigonopsis variabilis)中的daao基因及nptII (對照)基因為標誌基因,利用農桿菌轉移法將pCDAD及pRDAD等質體之上述基因轉移到甘藍、水稻與小白菜培植體。經D-alanine 或kanamycin篩選並誘導、再生成植株。轉殖植株以PCR分析之結果顯示,daao及nptII等選標誌基因已存在於轉殖甘藍、水稻與小白菜之基因組中。本研究同時將gus、daao及/或aadA基因,利用基因槍轉移法將pMT91-GD及pMT91-GDA等質體轉移到小白菜葉綠體中。經D-alanine 或spectinomycin篩選並誘導、再生成植株。轉殖植株以PCR及RT-PCR分析之結果顯示,gus、daao及aadA等基因已存在於轉殖T1及T2小白菜之基因組中,並表現daao mRNA。本研究顯示D-alanine的篩選系統在農桿菌介導法及基因槍轉移法的植物基因轉殖是可行的,並已初步完成建立小白菜之D-alanine篩選系統。由於小白菜培植體對spectinomycin及kanamycin之忍受毒害濃度的高敏感性,因此發展D-alanine的篩選系統,在植株外觀的篩選比spectinomycin及kanamycin篩選的優點更顯而易見。 本論文第四章以D型胺基酸氧化酵素(daao)及D-型胺基酸消旋酵素(D-amino acid racemase, alaR)基因作為篩選標誌基因,將轉穀氨醯胺酵素基因(tga)轉移至小白菜之葉綠體中。本研究之目的為探討利用小白菜葉綠體為非抗抗生素篩選標誌基因的生物反應器,生產轉穀氨醯胺酵素的可行性,以提高小白菜之經濟效益。本研究將自放線菌Streptomyces netropsis中篩選出的轉榖氨醯胺酵素基因(tga),所構築到小白菜葉綠體基因轉殖之八種載體pMT91-GTA、pMT91-GPA、pMT91-ETA、pMT91-EPA、pMT91GPsDA、pMT91GPsRA、pMT91EPsDA和pMT91EPsRA,利用基因槍法轉殖到'台農3號'小白菜之本葉的葉綠體。培植體經100~300 mg/L D-alanine或1~30 mg/L spectinomycin漸進篩選及誘導再生,目前已獲得T3轉殖子代。T0~T3 葉片經PCR分析之結果顯示,tga基因已存在於轉殖小白菜之葉綠體基因組中,且部份alaR及aadA等篩選標誌基因已被惕除,不存在小白菜的葉綠體基因組中。轉殖小白菜之葉片可偵側到TGA酵素活性,其中有二個轉殖系之TGA酵素活性較未轉殖對照組增加150~200%。本研究之結果顯示以daao及alaR等非抗抗生素篩選標誌基因之葉綠體基因轉殖技術,應用在小白菜葉綠體轉殖植株作為生物反應器,以生產轉穀氨醯胺酵素是可行的。 本論文第五章之研究目的為探討利用無篩選標誌基因之葉綠體基因轉殖系統,培育出抗病小白菜之可行性。本研究將分離自Xanthomonas fragariae (草莓角斑病菌)菌株的類似噬菌體 (phage XF)的溶菌酶 (lys),並構築到甘藍葉綠體基因轉殖之四種載體 (pMT91-GLsA、pMT91-ELsA、pMT91F-GLsA、pMT91F-ELsA),利用基因槍法轉殖到'台農3號'小白菜之本葉的葉綠體。培植體經1~30 mg/L spectinomycin漸進篩選及誘導再生,目前已獲得T1轉殖子代。T0及T1葉片經PCR及RT-PCR分析之結果顯示,lys基因已存在於轉殖之小白菜的葉綠體基因組中,並可表現lys mRNA。本研究初步結果顯示,利用葉綠體基因轉殖系統,轉殖溶菌酶基因 (lys)到小白菜,是可行的。
URI: http://hdl.handle.net/11455/89165
其他識別: U0005-2108201517500700
文章公開時間: 2018-08-25
Appears in Collections:園藝學系

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