臺灣博碩士論文加值系統

本研究自SBS轉錄體資料庫中選取一水稻花藥專一表現之基因（Os08g43240），在其基因5’端上游設計專一性引子，以PCR方式擴增1.5 kb啟動子序列，命名為P19啟動子。將啟動子序列自5’端刪除為1.2 kb、0.9 kb、0.6 kb、0.3 kb片段，分別接上含有報導基因GUS的C1301載體，轉殖於水稻及菸草當中，並以組織化學染色法分析轉殖兩種植物中P19啟動子表現的組織專一性。結果發現，P19啟動子可以驅動GUS基因在水稻及菸草花藥組織中大量表現。根據序列刪減分析，推測兩者的主要花藥發育調控序列不同，水稻位於-300 bp至-600 bp片段中；菸草則位於-300 bp片段之內。另外，在-1500 bp至-2000 bp片段則可能存在物種專一性的調控序列，調控基因表現與否。除了花藥組織之外，P19基因尚可驅動GUS基因於水稻的內穎、外穎、柱頭以及鱗被表現；菸草也可在花萼、花瓣、柱頭、花柱、花絲、葉片等組織偵測到GUS活性。水稻在內外穎、鱗被的染色結果分別與菸草花萼、花瓣的結果相似；雌性生殖器官也得到相似的表現結果，GUS基因表現於柱頭而不在子房表現，顯示P19啟動子在兩植物中對於上述器官的發育調節機制類似。較不一致的基因表現出現在葉片組織，水稻的葉片除了600 bp片段啟動子之轉殖株葉緣受刻傷逆境誘導之外，其他均無明顯GUS表現；菸草的葉片則大多有GUS表現，顯示單雙子葉植物的葉片發育調控機制不同。綜合以上結果，P19啟動子主要驅動基因表現在花藥中，未來期望將P19啟動子應用於轉位子的基因釣取系統當中，藉此增進基因釣取系統的生殖細胞專一性轉位。

In this study, a putative anther-specific promoter was cloned from a rice anther-specific gene (Os08g43240) based on SBS transcription database. Specific primers were designed to clone 1.5 kb upstream sequences of the target gene by PCR. The amplified putative promoter was named P19 promoter. Series deletion was conducted from 5’ end of the promoter to acquire 1.2 kb, 0.9 kb, 0.6 kb and 0.3kb promoter fragments, which were individually ligated to GUS reporter gene in C1301vector and then introduced into rice and tobacco. Histochemical analysis was conducted to characterize the expression patterns of the series deletion promoters. The result of staining showed that P19-GUS expression was observed in anther, lemma, palea, stigma and lodicule of transgenic rice, while in anther, filament, sepal, petal, stigma and style of transgenic tobacco. The deletion analysis showed that the regulatory sequence of anther may be located in the region between -300 and -600 bp in transgenic rice and inside -300 bp in transgenic tobacco. In addition, a species-specific regulatory sequence of anther-specific expression may be located between -1500 and -2000 bp of P19 promoter. The GUS expression patterns in lemma, palea and lodicule of transgenic rice were similar to those in sepal and petal of transgenic tobacco. The same phenomena were also observed in pistils of transgenic rice and transgenic tobacco, which indicated that the developmental mechanisms of these tissues may be directed by P19 promoter in similar ways. Nevertheless, the GUS activities were detected in most all transgenic tobacco leaves while the activities were detected only by wounding treated transgenic rice leaves. The leaves staining results showed that the regulatory mechanism may be different between monocot rice and dicot tobacco. In conclusion, P19 promoter directs gene expression mostly in anthers, and it is considered to be a potential promoter to enhance germinal transposition in transposon gene tagging system.

致謝 i
中文摘要 ii
英文摘要 iii
圖目錄 v
表目錄 vi
附錄目錄 vii
縮寫字對照表 viii
前言 1
文獻回顧 2
實驗設計與流程 15
材料方法 16
結果 27
討論 31
未來展望 35
參考文獻 36

圖目錄
圖 一、P19-C1301序列刪減系統之構築流程 49
圖 二、以限制酵素分析P19啟動子序列插入方向 50
圖 三、以PCR方式檢測P19-C1301序列刪減系統水稻及菸草轉殖系植株 51
圖 四、P19-C1301序列刪減系統之水稻轉殖系花藥GUS染色結果 52
圖 五、P19-C1301序列刪減系統之水稻轉殖系葉片GUS染色結果 53
圖 六、P19-C1301序列刪減系統之菸草轉殖系花藥GUS染色結果 54
圖 七、P19-C1301序列刪減系統之菸草轉殖系花苞GUS染色結果 55
圖 八、P19-C1301序列刪減系統之菸草轉殖系葉片GUS染色結果 56

表目錄
表 一、P19-C1301序列刪減系統所獲得之轉殖株情形 57
表 二、P19-C1301序列刪減系統於水稻之表現情形 58
表 三、P19-C1301序列刪減系統於菸草花藥之表現情形 59
表 四、P19-C1301序列刪減系統於菸草其他組織之表現情形 60

附錄目錄
附錄 一、Os08g43240基因於水稻花藥發育及各組織轉錄體分析結果 61
附錄 二、P19-C1301序列刪減系統之水稻轉殖系花藥GUS染色結果 62
附錄 三、P19-C1301序列刪減系統之菸草轉殖系花藥GUS染色結果 63
附錄 四、本試驗使用之引子一覽表 64
附錄 五、本試驗使用之培養基配製方法 65
附錄 六、本試驗使用之藥品來源 69

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