臺灣博碩士論文加值系統

植物運用維管束系統來面對環境中多樣的刺激，以調節自身的生長發育。近來的研究證實， 植物在葉部整合光週期的變化並利用維管束系統長距離輸送 FLOWERING LOCUS T (FT) 至頂芽，促進植物的開花。因此，FT 被認為是追尋已久的開花素 (Florigen)。然而，影響植物開花的因素除了光週期外，證據顯示尚有其他基因在植物開花的路徑中扮演系統性的調控因子。我們分析青花菜收取之篩管液，企圖找出潛在的系統性開花調控因子。利用阿拉伯芥生物晶片 (Arabidopsis GeneChip, ATH1-121501) 及反轉錄聚合酶連鎖反應 (RT-PCR) 的分析方法，從青花菜篩管液中鑑定出 FVE 與 AGAMOUS-LIKE 24 (AGL24) 兩個與開花調控有關的 RNA 分子。酵素學上的分析顯示篩管液中的 FVE 與 AGL24 的 RNA 帶有 5'-cap 結構及 polyadenylation tail。進一步，我們以野生型阿拉伯芥為接穗，嫁接至 FVE 或 AGL24 轉殖株之砧木上。利用 RT-PCR 的方式，可由野生型接穗偵測出 FVE 或 AGL24 轉殖基因之 RNA。顯示 FVE 與 AGL24 RNA 可在阿拉伯芥中進行長距離運送。綜合以上結果我們推論，FVE 與 AGL24 可能藉由長距離運送的方式，進而調控植物開花。

Plants take advantage of the vascular system to operate environmental stimulates for fine-tuning their developmental programs. Recent evidence shows that the FLOWERING LOCUS T (FT) protein is the long-sought-after florigen that integrates the photoperiod variation perceived in the leaves. However, evidence also supports that other yet-to-be identified systemic regulators participate in floral induction. To this end, we investigated phloem exudates from excised broccoli (Brassica oleracea) inflorescences. Microarray and RT-PCR analyses revealed that at least two RNAs of floral regulators, FVE and AGAMOUS-LIKE 24 (AGL24), are present in the phloem sap. Enzymatic analysis demonstrated that the phloem-sap RNAs contain a 5' cap and a polyadenlylation tail, which suggests that phloem sap contains typical mRNAs. Arabidopsis grafting experiments were used to test whether these RNAs move long distance along the phloem translocation stream. Consistent with previous reports, Arabidopsis transformants expressing FVE and AGL24 displayed an early flowering phenotype. When wild-type scions were grafted onto P35S-FVE or P35S-AGL24 transformant stocks, the RNAs of transgenic FVE and AGL24 were detected from the wild-type scions. Thus, both FVE and AGL24 RNAs can move long distance across the graft union. Our data support the notion that multiple systemic floral regulators may participate in floral regulation.

前言 …………………………………………………………………………. 1
材料與方法 …………………………………………………………………. 4
植物材料 ……………………………………………………………… 4
青花菜篩管液收集與 RNA 的萃取 …………………………………... 6
篩管液中蔗糖含量測定 …………………………………………….... 7
阿拉伯芥生物晶片(ATH1-121501, Affymetrix) 的雜合 (GeneChip hybridization) 與資料分析 …………………………………………… 8
校正程式 (probe masking files) 導入與分析方法 …………………… 9
聚合酶連鎖反應所需之基因引子對的搜尋與設計 ………………… 10
反轉錄聚合酶連鎖反應 (RT-PCR) ………………………………….. 11
mRNA decapping 檢測法 …………………………………………….. 12
質體 DNA 的構築 ……………………………………………………. 13
農桿菌基因轉殖 ……………………………………………………… 13
阿拉伯芥開花枝嫁接法 ……………………………………………… 14
結果 ………………………………………………………………………… 16
篩管液的收取與定性 ………………………………………………… 16
阿拉伯芥生物晶片分析 ……………………………………………… 17
青花菜篩管液基因群、維管束基因群與阿拉伯芥葉子基因群之比較 21
篩管液基因群分布在每個功能類別中 ……………………………… 21
青花菜篩管液中的 RNA 具有 5’ cap 與 polyadenylation tail ………. 22
青花菜篩管液中可偵測出與開花訊息調控路徑相關的 RNA 分子 .. 23
FVE 與 AGL24 RNA 在阿拉伯芥中會進行長距離運送 ……………. 24
許多有關植物訊息調控的路徑也有 RNA 分子存在青花菜篩管液中 . 25
討論 ………………………………………………………………………… 26
參考文獻 …………………………………………………………………… 31

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