苦瓜質脂結合蛋白基因 McPAP1 之功能分析及胡瓜基因轉殖系統之建立

Functional Analysis of Plastid-lipid-associated Protein Gene McPAP1 from Bitter Gourd and Establishment of Cucumber Transformation System

陳君琳(Chun-Lin Chen)

指導教授 ： 杜宜殷

共同指導教授 ： 黃鵬林(Pung-Ling Huang)

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10392

摘要

為瞭解苦瓜 (Momordica charantia L.) 質脂結合蛋白 (plastid lipid-associated protein) 之基因功能，本研究利用菸草 (Nicotiana tabacum L.) 作為轉殖材料，以分析苦瓜質脂結合蛋白基因 McPAP1 之啟動子活性及功能分析，結果顯示 McPAP1 啟動子活性於菸草轉殖株幼苗時期全株表現，而開花時期則專一性表現於生殖器官。誘導試驗顯示 McPAP1 啟動子於菸草轉殖株幼苗地上部與地下部具有顯著差異，於地上部可受到 BA、ABA、ACC 和 SA 以及創傷和 37℃ 黑暗誘導，受 GA 抑制活性表現；啟動子活性表現於地下部則受到 IAA、MeJA、高溫黑暗和乾旱誘導，受 BA 和 SA 抑制活性表現。過量表現 McPAP1 之菸草轉殖株具有植株高、花苞發育時間短、開花成熟期一致、平均節間長、葉基部大花朵數目多，且於乾旱和高光低溫環境耐受性較未轉殖株高等外表型，並且提高類胡蘿蔔素(carotenoid) 之貯存，但與類胡蘿蔔素生合成相關基因表現無關；綜合以上結果顯示，McPAP1 可能藉由穩定質粒結構，進而累積類胡蘿蔔素含量及穩定花器形成，由此推測 McPAP1 於營養生長階段與植株生長發育相關，而於生殖生長階段與花器發育具相關性。另外，本研究進一步建立胡瓜癒傷組織之再生及轉殖系統，以農桿菌媒介法將過量表現、默化表現以及蛋白質定位等構築轉殖至黃色鬆散之胡瓜癒傷組織，結果顯示以濃度 OD600 1.0 之農桿菌菌液、添加 100 μM AS、經超音波震盪 30 秒、進行感染 12 小時、共培養 48 小時之處理具較高轉殖率 94.80%。經由添加 0.1 mg•L-1 NAA、2 mg•L-1 Kinetin 以及 200 mg•L-1 kanamycin 之 MS 培養基同時進行篩選與再生，待轉殖細胞發生不定芽以獲得轉殖株。

關鍵字

基因過量表現 ； 類胡蘿蔔素 ； 花器形成 ； 基因轉殖 ； 不定芽誘導

並列摘要

To understand the function of plastid lipid-associated protein gene McPAP1 from bitter gourd, promoter activity and overexpression in tobacco transformants were analyzed. McPAP1pro::GUS transgenic tobacco showed GUS activity in the whole plant during seedling development, and specifically expressed in flower. Results of plant growth regulator and stress treatments indicated that the differential expression of McPAP1 between upper-ground part and roots. BA, ABA, ACC, SA, wounding and 37℃/dark induced McPAP1 transcription in the upper-ground, but part of seedling GA reduced the gene expression . IAA, MeJA, drought and 37℃/dark induced McPAP1 transcription in the roots, but BA and SA reduced the gene expression. McPAP1-overexpressed tobacco showed longer stem, earlier flowering, shorter maturity period of flowers, longer average internode, wider leaf base, more flower number, and more tolerance under drought or 180 μmol•m-2/8℃. Transgenic tobacco contained higher content of carotenoids, but gene expression of carotenoid-related showed no difference with the untransformed plant. McPAP1 probably accumulates carotenoids and assists flower maturation by stabilizing plastid structure. It might be involved in plant vegetative growth and flower organ differentiation. Moreover, plasmids for overexpression, silencing and protein localization were introduced into cucumber callus by Agrobacterium-mediated transformation. Using Agrobacterium cells whose concentration at 1.0 OD600 and supplementing 100 μM AS. The highest transformation efficiencies 94.80 % were obtained when calli subjected to a combination of sonication for 30 seconds followed by 12 hours of infection, and 48 hrs of cocultivation. After transformation, cucumber calli were cultured on MS medium supplemented with 0.1 mg•L-1 NAA, 2 mg•L-1 Kinetin, and 200 mg•L-1 kanamycin for selection and regeneration via adventitious shoots to whole plant.

並列關鍵字

gene overexpression ； carotenoids ； flower development ； transfotmation ； adventitious shoots induction

參考文獻

王瑞婷. 2010. 苦瓜質脂結合蛋白基因 McPAP1 之啟動子活性分析. 國立台灣大學園藝學系碩士論文.

江筱慧. 2008. 苦瓜質脂結合蛋白 cDNA 之選殖與分析. 國立台灣大學園藝學系碩士論文.

游沐慈. 2010. 苦瓜質脂結合蛋白基因 McPAP1 之功能分析及蛋白質定位. 國立台灣大學園藝學系碩士論文.

賈儒珍. 2005. 苦瓜性別表現相關蛋白質之研究. 國立台灣大學園藝學系碩士論文.

Amoah, B.K., H. Wu, C. Sparks, H.D. Jones. 2001. Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue. J Exp. Bot. 52: 1135-1142.