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研究生:陳怡潔
研究生(外文):I-Chien Chen
論文名稱:從土壤多源基因體中篩選出離胺酸消旋酵素並以離胺酸消旋酵素基因作為植物基因選殖的篩選標記
論文名稱(外文):Isolation and Characterization of a Novel Lysine Racemase Gene from a Soil Metagenomic Library and Utilization lyr as a Selectable marker for plant transformation
指導教授:許文輝許文輝引用關係
學位類別:博士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:102
中文關鍵詞:離胺酸消旋酵素土壤多源體基因庫篩選標記
外文關鍵詞:lysine racemasemetagenomic libraryselection marker
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Racemase具有生物轉換特性,催化天然及非天然胺基酸之旋光性,轉換質子的移動,產生不同旋光性胺基酸產物。本研究利用土壤多源體基因庫,搭配高複製套數的載體以及lysine營養缺陷菌株 Escherichia coli BCRC 51734,藉由功能性互補的方式,搭配D-lysine為特定氮源,選殖出離胺酸消旋化酶 (lyr) 基因。lyr基因由393個胺基酸所組成,分子量為42.7 kDa。生物資訊分析顯示,Lyr蛋白C-端 (殘基 188-393)與E. coli K12 argC 基因N-acetyl-γ-glutamyl- phosphate reductase (NAGPR)蛋白的N-端 (殘基1-206)具有100 %的相似度。而Lyr蛋白的N-端 (殘基 1-187)與Deinococcus radiodurans ATCC 13939的N-acylamino acid racemase (NAAAR)蛋白的N-端 (殘基 1-187),則具有98.4 %的相似度。然而純化後的Lyr蛋白,卻不具有NAGPR以及NAAAR的酵素活性。分析Lyr酵素活性,其最適反應溫度及pH值分別為30oC與pH 8.0,若在反應中分別加入Co2+、Mg2+ 和Mn2+等二價金屬離子,則可提高酵素的反應活性。以ICP spectrometer分析Lyr蛋白,顯示每一莫耳的二具體蛋白分子中含有0.77莫耳的Zn2+金屬離子。此酵素具有很嚴格的基質專一性,只對lysine具有消旋化酶的催化活性,催化D型或L型lysine之消旋化作用,Lyr對於L型或D型lysine的specific activity分別為3.61 U/mg 及1.68 U/mg,kcat 值分別為0.085±0.003 以及0.036±0.002 min-1。本研究是首次從土壤多元基因體中分離選殖出lyr基因,並報導其基因序列。本研究中,利用lysine racemase (lyr)的生物轉換特性,以L-lysine作為篩選劑,作為植物基因轉形時之篩選標記,並成功應用於菸草及阿拉伯芥這兩種模式植物中。在植物對於胺基酸敏感性的研究中,發現植物對於L-lysine相當敏感,反之,對於D-lysine則無此現象。因此利用消旋化酶的生物轉換特性,將L-lysine轉換成D-lysine作為植物生長的氮源。發現,在Arabidopsis中利用lyr作為篩選標記,比傳統上使用kanamycin篩選的轉形效率高2倍,且轉形後的所得到的轉殖植物,其發芽速度、植物體質量、葉片數目及根的長度上,與野生型植物相比較並無顯著差異。另一方面,Lyr 蛋白在轉基因菸草植物中的表現活性為0.77 to 1.06 mU/mg protein,此外,比對轉殖菸草與野生型菸草的內生性胺基酸濃度,僅aspartic acid的濃度受到lyr表現的影響而改變,顯示,此基因對於內生性胺基酸的代謝之影響非常有限。值得注意的是,研究中發現植物的aspartate kinases可區別lysine的旋光性,僅受到L-lysine的回饋調控。本研究為首次報導利用lyr基因取代抗藥性基因,作為植物基因轉形時之篩選標記,使用於模式植物-Nicotiana benthamiana Domin. 及Arabidopsis,成功的獲得基因轉殖植物,此篩選標記未來將可廣泛被使用於D-lysine或L-lysine敏感的植物。
A lysine racemase (lyr) gene was isolated from a soil metagenomic library by functional complementation using Escherichia coli BCRC 51734, a lysine auxotrophic mutant, as host cell and D-lysine as selection agent. This lyr gene contained an open reading frame encoding a protein composed of 393 amino acids with a molecular mass of about 42.7 kDa. Bioinformatics analysis revealed that the C-terminal portion (residues 188-393) of lysine racemase (Lyr) showed 100% amino acid identity to the N-terminal portion (residues 1-206) of N-acetyl-γ-glutamyl- phosphate reductase (NAGPR) encoded by E. coli K12 argC gene and the N-terminal portion (residues 1-187) of Lyr displayed 98.4% identity to N-terminal portion (residues 1-187) of N-acylamino acid racemase (NAAAR) from Deinococcus radiodurans ATCC 13939. However, no NAGPR and NAAAR activities were found for purified Lyr. The temperature and pH optimal for the enzyme activity of the Lyr protein were determined as 30oC and pH 8.0, respectively. The enzyme activity was enhanced significantly by metal ions Co2+, Mg2+ and Mn2+. The dialyzed Lyr contained 0.77 mol of Zn2+ per mol of the dimeric enzyme as revealed by the ICP spectrometer assay. The enzyme exhibited higher specific activity toward lysine (3.61 U/mg protein versus 1.68 U/mg protein) as well as a higher kcat (0.085±0.003 versus 0.036±0.002 min-1) in the L-lysine to D-lysine direction as compared to the reversible reaction. This is the first report of cloning, purification and characterization of a novel lysine racemase gene from a soil metagenome.
In this study, a non-antibiotic based selection system using L-lysine as selective agent and the lysine racemase (lyr) as selectable marker gene to develop the transgenic plants in two model plant species viz., tobacco and Arabidopsis was established. In preliminary experiments showed that several plant species are sensitive to L-lysine, while D-lysine supports their growth. Therefore, we used Lyr to catalyze the racemization of L- to D-lysine which would be used by transgenic plants as nitrogen source. The efficiency of transformation was approximately two-fold higher than kanamycin selection in Arabidopsis. Transgenic tobacco and Arabidopsis plants selected on L-lysine exhibited normal growth characteristics as that of wild-type plants and grew vigorously upon transfer to soil. The specific activity of Lyr in transgenic tobacco plants selected on L-lysine ranged from 0.77 to 1.06 mU/mg protein, whereas no activity was virtually detectable in wild-type plants. In addition, the composition of the free amino acids, except aspartic acid, was not affected by the expression of lyr in the transgenic tobacco plants suggesting very limited interference with endogenous amino acid metabolism. Interestingly, our findings also indicated that the plant aspartate kinases may possess an ability to distinguish the enantiomers of lysine for feedback regulation. This is the first report of a novel selection marker for transformation in which lyr gene instead of drug-resistant gene is used as selectable marker in the genetic transformation of two model plant species viz., Nicotiana benthamiana Domin. and Arabidopsis using L-lysine as selection agent.
目錄
第一章 : Isolation and characterization of a novel lysine racemase from a soil
metagenomic library
英文摘要-------------------------------------------------------2
中文摘要-------------------------------------------------------3
前言
一. 土壤多元基因體----------------------------------------------------4
二. 基因庫之篩選方式----------------------------------------------------5
三. 旋光性胺基酸----------------------------------------------------6
四. 胺基酸消旋酵素-----------------------------------------------7
五. 研究目的---------------------------------------------------9
材料方法
一. 化學藥品--------------------------------------------------------10
二. 質體、菌株、酵素及培養基--------------------------------------10
三. E. coli質體DNA的抽取----------------------------------------------10
四. DNA片段的回收及純化--------------------------------------------10
五. 染色體DNA的抽取----------------------------------------------11
六. 引子的設計-------------------------------------------------------11
七. 聚合酵素鏈索反應------------------------------------------------11
八. DNA黏合反應----------------------------------------------------12
九. 土壤樣品DNA的萃取----------------------------------------------12
十. 土壤多元性基因庫之建立--------------------------------------------13
十一. lyr基因之篩選----------------------------------------------13
十二. lyr基因之定序分析------------------------------------------13
十三. lyr基因在E. coli的表現----------------------------------13
十四. 蛋白質之定量---------------------------------------------------14
十五. 蛋白質的電泳分析--------------------------------------------15
十六. Lyr酵活性分析-----------------------------------------------16
十七. 選殖E. coli argC基因---------------------------------------16
十八. 選殖D. radiodurans ATCC 13939 naaar基因-------------------------17
結果
一. 由土壤多元基因體選殖出lyr基因-----------------------------------------------19
二. Lyr蛋白的表現、純化及生化特性-----------------------------------------------20
三. NAGPR及NAAAR蛋白的表現、純化及生化特性-------------------------------------21
討論------------------------------------------------------------------23
參考文獻-------------------------------------------------------------27
圖表------------------------------------------------------------------------36

第二章 : Lysine racemase: a novel and efficient non-antibiotic selectable marker for
plant transformation
英文摘要---------------------------------------------------------------------49
中文摘要--------------------------------------------------------------------50
前言
一. 生物技術及基因工程技術----------------------------------------------------51
二. 植物基因轉殖技術--------------------------------------------------------51
三. 非載體媒介轉移法-------------------------------------------------------52
四. 載體媒介轉移法---------------------------------------------------53
五. 基因改造作物及其生物安全性問題--------------------------------------------55
六. 模式植物------------------------------------------------------------------57
七. 研究目的------------------------------------------------------------------58
材料方法
一. 化學藥品------------------------------------------------------------60
二. 質體、菌株、酵素及培養基-----------------------------------------60
三. 植物材料------------------------------------------------------------------60
四. 植物之種植------------------------------------------------------------------60
五. 重組DNA技術-----------------------------------------------------------61
六. pBI-lyr與pBI-km-lyr載體之構築--------------------------------------------61
七. 農桿菌勝任細胞之製備---------------------------------------------------61
八. 電穿孔方式將轉殖載體轉移入農桿菌-------------------------------------------62
九. 阿拉伯芥之基因轉殖及篩選----------------------------------------------62
十. 菸草之基因轉殖及篩選---------------------------------------------------63
十一. 植物染色體DNA之抽取------------------------------------------63
十二. 轉殖植物之初步確認-------------------------------------------63
十三. DNA探針的製備----------------------------------------------64
十四. 南方轉漬雜交法------------------------------------------------64
十五. 菸草蛋白之萃取---------------------------------------------------64
十六. 蛋白質膠體電泳分析-------------------------------------------65
十七. 西方轉漬雜交法-----------------------------------------------------66
十八. Lyr酵素活性分析---------------------------------------------66
十九. 胺基酸濃度分析-----------------------------------------------67
結果
一. 植物對胺基酸的利用----------------------------------------------68
二. 菸草轉殖株的篩選------------------------------------------------69
三. 阿拉伯芥轉殖株的篩選---------------------------------------------70
四. Lyr蛋白在轉基因植物中的表現及活性分析------------------------71
五. lyr基因在阿拉伯芥轉殖株的遺傳率--------------------------------71
六. 轉基因植物之胺基酸濃度變化--------------------------------------72
討論--------------------------------------------------------------------------73
參考文獻-------------------------------------------------------------76
圖表--------------------------------------------------------------------------81






















圖表目錄
第一章: Isolation and characterization of a novel lysine racemase from a soil
metagenomic library
表一 本實驗所使用的菌株和質體----------------------------------------------------36
表二 本實驗所使用的引子------------------------------------------------------38
表三 金屬螯合劑、金屬離子及化合物對Lyr活性的影響-------------------------39
表四 不同重組蛋白的酵素活性分析-------------------------------------------41
圖一 從土壤多源體基因庫所篩選出的lyr基因之核甘酸與胺基酸序列-----42
圖二 圖示Lyr, NAGPR及NAAAR蛋白之間的相似區域--------------------43
圖三 利用洋菜膠電泳分析及SDS-PAGE分析lyr基因的表現--------------44
圖四 pH值對Lyr活性的影響---------------------------------------------45
圖五 溫度及熱穩定度對Lyr活性的影響----------------------------------------46
圖六 Lyr對於催化L-lysine轉換成D-lysine之轉換率------------------48

第二章: Lysine racemase: a novel and efficient non-antibiotic selectable marker for
plant transformation
表一 本實驗所使用的菌株和質體----------------------------------------------------81
表二 本實驗所使用的引子------------------------------------------------------83
表三 阿拉伯芥轉基因植物利用不同篩選系統之轉形效率------------------------84
表四 比較野生型及轉機因阿拉伯芥植株之表型------------------------------------85
表五 lyr基因在T2轉基因阿拉伯芥轉殖株的遺傳率---------------------86
表六 野生型及轉基因菸草植株中游離胺基酸之濃度------------------------87
圖一 雙偶型載體pBI-lyr及pBI-Km-lyr之構築---------------------------------88
圖二A菸草種子種植於含有lysine的MS培養基之生長情形-------------------------89
圖二B菸草葉片於含有不同濃度的L-lysine的MS培養基之存活率-------------------90
圖三A阿拉伯芥種子種植於含有lysine的MS培養基之生長情形-----------------------91
圖三B阿拉伯芥種子於含有不同濃度的L-lysine的MS培養基之存活率-------------92
圖四 不同種子種植於含有L-lysine的MS培養基之生長情形----------------------------93
圖五 利用L-lysine作為篩選劑,篩選轉基因菸草植物------------------------------------94
圖六 利用洋菜膠電泳分析轉基因菸草植株染色體DNA中的lyr基因---------------95
圖七 利用南方轉漬雜交法分析菸草轉殖株的染色體DNA---------------------------96
圖八 阿拉伯芥轉殖株的篩選---------------------------------------------------97
圖九 利用洋菜膠電泳分析轉基因阿拉伯芥植株染色體DNA中的lyr基因-----------98
圖十 利用南方轉漬雜交法分析菸草轉殖株的染色體DNA------------------------------99
圖十一 利用西方轉漬雜交法分析lyr基因在T1菸草轉殖株及T2阿拉伯芥轉殖株的表現-------------------------------------------------------------------------------------------100
圖十二 利用HPLC分析菸草轉殖株的Lyr酵素活性-------------------------------------101
圖十三 觀察野生型與轉基因阿拉伯芥的生長情形---------------------------------------102
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