Expression of green fluorescent protein in tobacco hairy roots

以三種不同建構之質體於毛狀根中表現綠色螢光蛋白質 ( GFP )。皆使用 CaMV 35S 啟動子來表現下游 gfp：表現於細胞質中的 pCAMBIA 1302、帶有訊息序列 ( signal sequence ) 的 pCSP、以及轉譯後 N 端帶有訊息序列，C 端帶有內質網停留序列 ( ER-retention sequence ) 的pCSP-KDEL。將以上三種質體分別放入農桿根群菌中，並以其感染菸草，得到三群帶有不同外源基因的毛狀根。挑選除菌後生長良好的毛狀根進行液態培養，經 PCR、螢光顯微鏡、螢光分光光度計確認其表現，並以 ELISA 方式測定 GFP 之表現量。接著我們從三群轉型株中分別挑出一株 ( 分別為 CY A3、SP F3、KDEL J3 ) 表現量高的毛狀根進行培養分析。發現隨著培養天數增加，GFP 可以穩定持續表現，並於胞內及培養基中累積。以上三種轉殖株培養28天後 GFP 表現量分別為 985.5、258.9、54.2 ng / 50 mL，而培養基中 GFP 則分別佔 21、33、40%。結果顯示，GFP表現量以CY轉型株較高，但有訊息序列設計之轉型株則可提升 GFP 外泌到培養基中的比例。之後取出毛狀根進行細胞壁溶解的實驗，結果顯示，CY A3、SP F3 及 KDEL J3 釋出 GFP 到培養基中的比例分別為 20%、27% 及 35%。推測外泌設計表現之 GFP 可能受到細胞壁之阻礙而影響其外泌效率。Expression efficiency of green fluorescent protein ( GFP ) in hairy root cultures of Nicotiana tabacum by different gene construction designs was compared. These gene constructions including gfp driven by CaMV 35S promoter ( pCAMBIA 1302 ), then further fused with signal sequence to the 5’ of gfp ( pCSP ), and then further fused with ER-retaintion sequence to the 3’ of gfp ( pCSP-KDEL ). Establishment of Nicotiana tabacum transgenic hairy roots was achieved by Agrobacterium rhizogenes-mediated transformation, PCR confirmation, fluorescence microscopy observation, Western blot of GFP and its ELISA assay. Clone of CY A3, SP F3 and KDEL J3 was selected from each design for GFP expression comparison. After 28-d cultivation, the expressed GFP of the three constructions was 985.5, 258.9 and 54.2 ng / 50 mL, respectively, and the ratio of GFP secreted was 21, 33, and 40%, respectively. Among the three constructions, our results reveal that gene construction without secretion designs possessed the highest GFP expression efficiency, although SP and KDEL designs showed higher ratio of GFP in the cultured medium. In the experiment of enzymatic lysis of transgenic hairy roots cell wall, the released GFP of three transgenic line CY A3, SP F3 and KDEL J3 was 20%, 27% and 35% of the total cytosol GFP , respectively. It suggests that secretion of GFP in the design of SP and KDEL might be hindered by cell wall layer.Abstract...............................I 中文摘要..............................II Abbreviation.........................III 中英文專有名詞對表....................IV Contents...............................V Contents of tables and figures.......VII Chapter I. Introductions...............1 Chapter II. Materials and Methods.....10 Chapter III. Results..................25 Chapter IV. Discussion................28 Tables................................35 Figures...............................38 References............................58 Appendix..............................6