Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36913
DC FieldValueLanguage
dc.contributor.advisor古新梅zh_TW
dc.contributor.advisorFuh-Jyh Janen_US
dc.contributor.advisor詹富智zh_TW
dc.contributor.author張惠如zh_TW
dc.contributor.authorChang, Hui-Juen_US
dc.date2005zh_TW
dc.date.accessioned2014-06-06T07:58:12Z-
dc.date.available2014-06-06T07:58:12Z-
dc.identifier.urihttp://hdl.handle.net/11455/36913-
dc.description.abstract植物在滲透逆境下,普遍以調節脯胺酸含量來回應環境變化,與脯胺酸生成代謝相關基因,包括三種生成酵素基因 ( pyrroline-5-carboxylate synthetase, P5CS; pyrroline-5- carboxylate reductase, P5CR; ornithine aminotransferase, OAT ) 與一種代謝酵素基因 ( proline dehydrogenase, PDH )。本實驗室先前研究指出乾旱處理可誘導菸草脯胺酸累積,同時也使P5CS與OAT基因表現升高,而PDH基因表現則被抑制。因此,本研究利用病毒誘導基因沉寂 ( virus-induced gene silencing, VIGS ) 策略,建立可同時抑制多個基因表現之系統,來抑制脯胺酸生成及代謝相關基因的表現量,以了解在缺水逆境下脯胺酸的累積是藉由何種路徑,並進一步探討P5CS與OAT扮演角色。由菸草 ( Nicotiana benthamiana ) 選殖此四個基因的部分片段及定序,並與已登錄於基因庫的番茄 ( Lycopersicon esculentum ) P5CS, P5CR, OAT, PDH基因分別進行核酸序列比對,在選殖到之約400 bp 之基因片段中至少有82.1% 的核酸序列相同性( nucleotide identity )。將這些基因片段反義股 ( antisense ) 構築於可利用於VIGS策略之菸草鑲嵌病毒 ( Tobacco mosaic virus, TMV ) 載體中。在單逢乾旱逆境時P5CS基因表現量在缺水24小時內大幅提升;OAT基因表現量在缺水36小時後,才有高於10倍的提升。為更進一步探討,遂以上述構築之TMV載體接種於菸草進行病毒誘導基因沉寂作用,試驗結果顯示。抑制P5CS基因表現對脯胺酸累積的影響較抑制OAT基因時大。另外,同時抑制P5CS, OAT及PDH基因時,P5CS與OAT基因抑制量較同時抑制P5CS與OAT基因少,脯胺酸合成效率相對增加,此情況下脯胺酸累積量較高並非歸因於PDH基因表現被抑制。再則,PDH基因的大量表現乃出現於乾旱回水的過程。而抑制或乾旱誘導P5CR基因表現量對於脯胺酸累積量並無明顯影響。根據結果推論,乾旱誘導脯胺酸累積應為調控脯胺酸合成速率的增加。P5CS與OAT基因的確皆參與乾旱誘導脯胺酸累積的情形,且在乾旱初期P5CS基因的貢獻較OAT基因大。因此在菸草葉片處於乾旱逆境時,P5CS基因是扮演第一反應角色。另一方面,利用VIGS系統針對單一或多個基因表現,探討乾旱逆境下菸草脯胺酸生合成代謝相關基因與脯胺酸累積之相互關係,所得與菸草在乾旱逆境下脯胺酸生合成代謝相關基因表現調控脯胺酸累積之結果相符。本論文建立以VIGS系統同時探討乾旱逆境時參與脯胺酸生合成代謝相關基因之表現及其可能意義,此一正面結果有利於以VIGS探討多基因功能之實際應用,在往後研究植物基因功能時可提供較全面性的探討及結論。zh_TW
dc.description.abstractMany plants synthesize and accumulate proline in response to osmotic stress. Four major proline metabolism related genes (PMRGs) that include three proline synthesis genes, pyrroline-5-carboxylate synthetase (P5CS), pyrroline-5-carboxylate reductase (P5CR) and ornithine aminotransferase (OAT), and one proline catabolism gene, proline dehydrogenase (PDH) regulate proline biosynthetic pathways in plants. We previously demonstrated that drought stress increased the proline content in tobacco (Nicotiana benthamian) leaves along with the increase of P5CS and OAT gene expression, and with the decrease of PDH gene expression. In this study, to characterize transcriptional regulation of the key proline enzymes in tobacco N. benthamiana, four PMRGs DNA fragments (around 400 bps each) were identified and cloned. The sequences of those gene fragments from tobacco shared high homologies with more than 82.1% nucleotide sequence identity to those of tomato available in Genbank. To assess the relative important role of PMRGs involved in proline metabolism during drought stress in the tobacco plants, those gene fragments were constructed into Tobacco mosaic virus (TMV) viral vector to establish a system that can suppress multiple genes expression simultaneously via virus-induced gene silencing (VIGS). The P5CS activity increased in the primary stage (24 hour treatment) of drought stress, but the OAT activity increased in the later stage (36 hour drought treatment). Suppression of the P5CS gene expression via VIGS increases proline accumulation more efficient than that of suppression of OAT gene expression. In addition, the gene expression of P5CR did not affect proline accumulation under both drought and VIGS, suggesting that P5CR gene does not play important role in drought-induced proline accumulation. On the other hand, the PDH gene activity increased in rewater and infection of TMV containing antisense RNA of P5CS, OAT and PDH on tobacco. The fact that PDH gene was suppressed under drought treatment is possibly benefit to proline accumulation, but is unlikely able to overcome the action of P5CS and OAT genes. Based on these results, we suggest that drought-induced proline accumulation was via regulating the increase of P5CS and OAT gene expression. Thus both ornithine and glutamate biosynthesis pathways contribute key roles to the drought stress-induced proline accumulation in the N. benthamiana plants. P5CS especially plays a major role for the proline accumulation in the early stage of drought stress. In addition, the VIGS system for suppressing multiple genes simultaneously developed in this study can not only be used to confirm the function of PMRGs but also study other metabolic pathway in the further.en_US
dc.description.tableofcontents中文摘要………………………………………………………………… 01 Abstract………………………………………………………………… 03 前人研究………………………………………………………………… 05 材料與方法……………………………………………………………… 14 試驗材料……………………………………………………………… 14 總量 RNA (total RNA) 之萃取……………………………………… 14 ornithine-δ-aminotransferase (OAT)、∆1-pyrroline-5-carboxylate synthetase (P5CS)、∆1-pyrroline -5-carboxylate reductase (P5CR) 及proline dehydrogenase (PDH) 基因片段之選殖…………………… 15 OAT引子對之設計…………………………………………………… 15 P5CS引子對之設計…………………………………………………… 15 P5CR引子對之設計…………………………………………………… 16 PDH引子對之設計…………………………………………………… 16 反轉錄聚合酵素鏈鎖反應 (reverse transcriptase polymerase chain reaction, RT-PCR)……………………………………………………… 17 基因片段選殖………………………………………………………… 18 OAT、P5CS、P5CR及PDH基因片段之定序及分析………………… 19 攜帶單一脯胺酸生合成代謝相關基因的VIGS載體之構築………… 19 大腸桿菌的轉形作用 (transformation)…………………………… 20 攜帶多個脯胺酸生合成代謝相關基因的VIGS載體之構築………… 21 生體外轉錄作用 (in vitro transcription )……………………… 22 酵素連結免疫吸附反應 (enzyme-linked immunosorbent assay, ELISA)…… 23 乾旱逆境之處理與葉綠素、蛋白質、MDA、脯胺酸含量測定………… 24 Malondialdehyde (MDA) 之檢測………………………………… 24 脯胺酸之檢測………………………………………………………… 25 葉綠素之檢測………………………………………………………… 26 蛋白質之檢測………………………………………………………… 26 同步定量聚合酶鏈鎖反應 ( real-time RT-PCR) …………………… 26 數據分析……………………………………………………………… 28 結果……………………………………………………………………… 30 選殖Ornithine aminotransferase (OAT) 之部份基因片段……… 30 選殖Pyrroline-5-carboxylate synthetase (P5CS) 之部份基因片段…31 選殖Pyrroline-5-carboxylate reductase (P5CR) 之部份基因片段……31 選殖Proline dehydrogenase (PDH) 之部份基因片段……………… 32 菸草在乾旱逆境下生理變化及脯胺酸生成代謝相關基因之表現… 33 脯胺酸生成代謝相關基因VIGS載體之構築……………………… 35 以TMV載體接種菸草進行VIGS及其在缺水逆境下的生理變化與脯胺酸生成代謝相關基因的表現……………………………………… 36 討論……………………………………………………………………… 39 參考文獻………………………………………………………………… 45 圖表……………………………………………………………………… 56zh_TW
dc.language.isoen_USzh_TW
dc.publisher農藝學系zh_TW
dc.title以病毒誘導基因沉寂策略進行菸草脯胺酸庫之代謝工程zh_TW
dc.titleMetabolic engineering of tobacco proline pool via virus-induced gene silencingen_US
dc.typeThesis and Dissertationzh_TW
Appears in Collections:農藝學系
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