菸草毛狀根之蛋白質體學研究

Proteomic study in hairy roots of Nicotiana tabacum L. var Wisconsin 38

楊舒晴(Shu-Ching Yang)

指導教授 ： 李昆達

共同指導教授 ： 陳逸然(Yet-Ran Chen)

國立臺灣大學/生命科學院/生化科技學系/碩士(2011年)

https://doi.org/10.6342/NTU.2011.02471

摘要

經根毛農桿菌 (Agrobacterium rhizogenes) 轉形的毛狀根具有生長快速與高次級代謝物生合成能力之優點，然而，調控機制卻尚未釐清。本研究從蛋白質體學角度切入，期望找出菸草 (Nicotiana tabaccum L. var Wisconsin 38) 毛狀根與一般根在蛋白質層次的表現差異，進而找出參與生長調控與刺及代謝物生產之蛋白質。利用聚合酶連鎖反應確認根毛農桿菌之 T-DNA 鑲嵌至菸草的染色體上。經根毛農桿菌轉形之菸草，其毛狀根之生長速度與尼古丁含量皆優於一般根。利用二維電泳結合液相層析質譜與二維液相層析質譜儀分析菸草一般根與經根毛農桿菌轉形之毛狀根總蛋白質，比對具差異性表現量的蛋白質點並鑑定蛋白質身分。我們發現在毛狀根中與根部發育相關之蛋白質，如參與轉錄作用、轉譯作用、蛋白質摺疊與組合、能量代謝、胺基酸與脂肪酸生合成、代謝物生合成等蛋白質於毛狀根中皆受到正調控。此外，與DNA 纏繞、蛋白質降解、氧化逆境平衡等蛋白質表現則受到負調控。此蛋白質體比較結果將有助於了解毛狀根生長快速與高次級代謝物生產力之調控機制，並益於未來毛狀根之應用。

關鍵字

根毛農桿菌 ； 菸草 ； 蛋白質體學 ； T-DNA ； 尼古丁

並列摘要

Hairy root is known for its rapid growth and high metabolite production ability. However, little is known about how the production of those metabolites is regulated in hairy roots. In this study, we aim from the proteomic view to study the change of protein expression patterns between hairy roots and normal roots, expecting to find proteins related to the rapid root growth and the production ability of secondary metabolites. The T-DNA insertion of hairy roots derived from Agrobacterium rhizogenes-mediated transformation of Nicotiana tabaccum L. var Wisconsin 38 was confirmed by PCR. Hairy roots not only grown faster but synthesized more nicotine than normal roots. Normal roots and hairy root clones were analyzed by two-dimensional electrophoresis coupled with LC-MS/MS and gel-free 2D-LC-MS/MS. Comparative analysis of the protein patterns indicated a number of proteins showed different accumulation levels relative to normal roots. We found that proteins participated in transcription, translation, protein folding and assembly, energy metabolism, amino acid and fatty acid synthesis, metabolite synthesis and those related to the root development were up-regulated in hairy roots. In addition, proteins involved in DNA packaging, protein degradation, and reactive oxygen (ROS) balance were down-regulated in hairy roots. The comparison of the proteome between the hairy roots and the normal roots will be helpful for revealing the unique regulation mechanism in hairy roots and benefiting the future application of the hairy roots.

並列關鍵字

Agrobacterium rhizogenes ； tobacco ； proteomics ； transfer-DNA ； nicotine

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