Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/52433
標題: Alanine Racemase as a Novel Selectable Marker for Plant Transformation and Elucidation of Mechanisms of D-amino Acid Toxicity in Plants
利用Alanine racemase做為植物基因轉殖的篩選標誌並釐清D-amino acid對植物具有毒性之機制
作者: 許文輝
關鍵字: 生物技術, 生物科學類
應用研究
Alanine racemase
丙胺酸消旋酵素
非抗藥性標記
阿拉伯芥
D 型胺基酸毒性
gek1 突變株
核酸微矩陣
non-antibiotic marker
D-alanine
Arabidopsis
D-amino acidtoxicity
gek1 mutants
DNAmicroarray
摘要: 植物基因轉殖需要適當的篩選標記,目前大多利用抗藥性與抗殺草劑基因作為基因重組的篩選標記,將這些基因使用於植物基因轉殖,可能會對人類或環境有不利的影響。有鑑於此,利用其他非抗藥性基因作為植物基因轉殖之正向篩選標記,有其必要性。近年來,雖然已發表數種非抗藥性正向篩選標記,但是這些篩選標記在商業應用上皆有所限制,雖然可成功的獲得轉殖株,惟能應用的植物種類有限,並易導致後代轉基因植物農藝性狀的改變。最近,我們從土壤多元體基因庫中選殖出lysine racemase 基因(Appl.Environ. Microbiol., 2009),並發現此基因可以取代抗藥性基因,以L-lysine 作為篩選劑,作為植物基因轉形時之篩選標記,應用於菸草及阿拉伯芥這兩種模式植物的基因轉殖(Plant Mol. Biol., 2009)。然而,此種篩選系統僅適用於對L-lysine 敏感的植物種類。現有的報告顯示,植物對於某些D 型胺基酸相當敏感,會造成植物生長抑制及其他生理上的失調。顯然,利用此種生理特性,可建立其他的非抗藥性的篩選系統,俾能廣泛的使用於各種植物的基因轉殖。除此之外,D 型胺基酸在動物的生理角色已有相當的研究,但對植物的毒性則尚未釐清。因此,本研究的主要目的在於:(1) 發展新型的非抗藥性篩選系統,作為植物基因轉殖之用,在非抗藥性標記方面,將以Corynebacterium glutamicumNCHU 87078 的alanine racemase 基因(alr)為正篩選標記,以D-alanine 作為篩選劑;(2) 利用模式植物Arabidopsis 為材料,以胺基酸轉運蛋白、胺基酸生合成相關基因...等為標的,由核酸微矩陣、即時RT-PCR 和基因產物生化特性分析,以及觀察alr 轉殖株經D-alanine 處理後之農藝性狀,來釐清D 型胺基酸對植物生長具有毒性之可能機制。三年研究計畫內容如下:第一年一、構築含有C. glutamicum NCHU 87078 alr 基因的植物表現載體, 轉形入Agrobacterium GV3101,進行Arabidopsis 之轉殖作用二、轉殖株篩選條件之最適化,並用含D-alanine 的培養基回收表現alr 基因的初代轉殖株三、增加T1 同質品系植株的數量四、以核酸微矩陣分析經D-alanine 處理或未處理的Arabidopsis 幼苗,了解全體基因表現的差異第二年一、選殖後的同質轉基因品系之遺傳、表型、生化及分子的特性分析二、分析表現alr 基因之轉基因植物的胺基酸之組成份三、利用具有毒性的D-alanine 來篩選gek1 突變株及gek1-overexpression 轉殖株,並觀察他們的胺基酸組成變化四、根據核酸微矩陣分析結果及即時聚合鏈鎖反應(real-time RT-PCR),推論deacylase酵素,胺基酸轉運蛋白,胺基酸生合成基因...等與D-alanine 毒性之間的關係第三年一、與D-alanine 毒性有關連性的基因產物之生化特性分析二、利用透射電子顯微鏡,觀察D-alanine 處理或未處理的Arabidopsis 植株之根部細胞結構之差異
Public concerns on the biosafety and regulatory issues over the use of antibiotic andherbicide-based selectable markers have necessitated the scientists to develop alternativeapproaches for transgenic plant selection. Although several kinds of positive selectablemarkers have been developed during recent years, the potential application of such markersfor commercial purposes is often limited by the facts that they are mostly sub-optimal inpractice and often produce undesirable consequences in plant phenotypes. Very recently, wehave successfully isolated a lysine racemase gene from soil metagenomic library (Appl.Environ. Microbiol., 2009) and demonstrated the utilization of lysine racemase as anon-antibiotic marker using L-lysine as selection agent for the transformation of Arabidopsisand tobacco (Plant Mol. Biol., 2009). However, the use of our system is limited tolysine-sensitive plants. Therefore, it is imperative to establish a novel non-antibiotic andregulatory-friendly marker system for wider use in the development of transgenic plants. Ithas been known that plants are highly sensitive to exogenously applied D-amino acids,resulting in the growth inhibition and other physiological disorders. This suggests thatD-amino acids generally may not be utilizable as sources of nitrogen for plants and interferewith plant metabolism. In contrast to considerable understanding of D-amino acids inmammalian physiology, the toxicity of D-amino acids to plants remains to be elucidated.This proposal is primarily aimed at the development of a novel non-antibioticselectable marker system for plant transformation and elucidation of possible mechanisms ofD-amino acid toxicity in plants using the model species Arabidopsis. For the marker systempart, the major focus will be to demonstrate the use of alanine racemase (alr) gene fromCorynebacterium glutamicum NCHU 87078 as a positive selectable marker and D-alanine asthe selection agent. For D-amino acid toxicity part, the main thrust would be to gain newinsights into the mechanisms of toxicity based on the studies of amino acid transporters,amino acid biosynthetic genes etc. through analyses of the data from microarray, real-timeRT-PCR, biochemical properties of gene products, and agronomic properties of transgenicArabidopsis.The research work will be planned for three years as follows:First grant year1. Construction of plant expression vector containing the alr gene from C. glutamicumNCHU 87078 and mobilization into Agrobacterium strain GV3101 for Arabidopsistransformation2. Optimization of selection conditions and recovery of primary transformants expressingthe alr gene on D-alanine medium3. Generation advancement of T1 plants to obtain homozygous transgenic lines4. To study the changes in global gene expression in the control and D-alanine treatedArabidopsis seedlings by microarray analysisSecond grant year1. Genetic, phenotypic, biochemical and molecular characterization of selectedhomozygous transgenic lines2. Analysis of endogenous amino acid composition in the alr-expressing transgenic plants3. Screening of gek1 mutants and gek1-overexpressing transgenic plants on toxic D-aminoacids and observation of differences in their amino acid profile4. To correlate deacylase, amino acid transporters and amino acid biosynthetic genes etc. toD-amino acid toxicity based on microarray data and real-time RT-PCRThird grant year1. Biochemical characterization of the gene products involved in D-amino acid toxicity2. Examination of ultrastructural changes/differences in the root cells of control andD-alanine treated Arabidopsis plants using transmission electron microscopy
URI: http://hdl.handle.net/11455/52433
其他識別: NSC99-2628-B005-005
文章連結: http://grbsearch.stpi.narl.org.tw/GRB/result.jsp?id=2103855&plan_no=NSC99-2628-B005-005&plan_year=99&projkey=PD9907-0691&target=plan&highStr=*&check=0&pnchDesc=%E5%88%A9%E7%94%A8Alanine+racemase%E5%81%9A%E7%82%BA%E6%A4%8D%E7%89%A9%E5%9F%BA%E5%9B%A0%E8%BD%89%E6%AE%96%E7%9A%84%E7%AF%A9%E9%81%B8%E6%A8%99%E8%AA%8C%E4%B8%A6%E9%87%90%E6%B8%85D-amino+acid%E5%B0%8D%E6%A4%8D%E7%89%A9%E5%85%B7%E6%9C%89%E6%AF%92%E6%80%A7%E4%B9%8B%E6%A9%9F%E5%88%B6
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