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研究生:陳信宇
研究生(外文):Chen Hsin-Yu
論文名稱:山藥貯藏蛋白質dioscorin基因序列與調控序列之選殖與探討
論文名稱(外文):Studies on the genomic and the regulatory sequence of the storage protein dioscorin genes of yam(Dioscorea alata L.)
指導教授:周微茂
指導教授(外文):Chou Wei-Mou
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:94
語文別:中文
論文頁數:80
中文關鍵詞:山藥 (Dioscorea alata L.)貯藏蛋白質基因熱非對稱交互PCR (TAIL-PCR)農桿菌滲入轉殖法
外文關鍵詞:yam(Dioscorea alata L.)storage protein genedioscorinTAIL-PCRagroinfiltration
相關次數:
  • 被引用被引用:2
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山藥(Dioscorea spp.)塊莖中主要的貯藏蛋白質為dioscorin。本研究根據NCBI Genbank中dioscorin A 的mRNA序列,設計數對引子進行巢狀PCR(Nested PCR)及熱非對稱交互PCR (Thermal asymmetric interlaced PCR, TAIL-PCR),從山藥(D. alata)的基因組 DNA中,選殖到四段彼此部份重疊的dioscorin 基因序列,得知全長的dioscorin基因含6個內插子, 7個外顯子。再進行內插子序列的選殖,6個內插子分別選殖到2種,2種,3種,3種,3種,4種,共得到17種不同的內插子序列。第三個與第四個內插子的9種組合中,由基因組 DNA確實選殖到6條序列,推測dioscorin基因家族中至少有6個dioscorin A基因家族成員。
本研究也利用TAIL-PCR,在山藥基因組 DNA中,選殖到一段491 bp的 dioscorin基因5’端之上游序列,包含dioscorin基因的轉譯起點5’端上游 466 bp,及5’-UTR 74 bp 的DNA序列。利用資料庫軟體進行序列比對及啟動子預測分析,發現具有啟動子以及其他轉錄因子的序列。
根據預測的功能活性區,建構7段5’端漸次序列刪除的片段,並分別構築融合GUS基因的質體,以農桿菌滲入轉殖法在菸草幼苗葉片中測試其短暫性表現,期能快速評估其轉錄活性,用以分析並確定其活性區域。
Dioscorin is the major storage protein in yam (Dioscorea alata L.)。In this study, four fragments of the genomic sequences of the storage protein dioscorin genes were cloned by nested PCR and TAIL-PCR(Thermal asymmetric interlaced PCR)with several primer pairs designed according to the dioscorin A mRNA sequences published in NCBI Genbank. The dioscorin gene should be comprised of 7 exons and 6 introns. In the intron assay, we cloned 17 different introns totally. The 1st intron has 2 different kinds, the 2nd has 2, the 3rd has 3, the 4th has 3, the 5th has 3, and the 6th has 3. We concluded that there are at least 6 members of dioscorin A in the dioscorin gene family according the 6 cloned fragments combined with the 3rd and the 4th different introns.
Furthermore, the 5’-upstream sequence of the yam storage protein dioscorin gene was isolated by TAIL-PCR from the genomic DNA. The product was 491 bp with the 466 bp upstream to the translation start site of the dioscorin gene and 74 bp 5’-UTR. After the sequence alignment and identification analysis in the database, the putative promoter and several putative cis-acting elements were predicted.
The sequence was used to generate 7 fragments of 5’ serial deleted sequence and fused with GUS gene of Ti-plasmid. The agroinfiltration method was applied for transient expression in the leaves of tobacco seedlings to rapidly examine its transcriptional activity and confirm the cis-acting elements .
目 錄
中文摘要Ⅰ
英文摘要Ⅱ
致 謝Ⅲ
圖表索引Ⅷ
第一章、前人研究1
第一節、塊莖貯藏蛋白質的研究1
第二節、山藥貯藏蛋白質dioscorin特性與活性4
第三節、塊莖貯藏蛋白質基因的調控與表現6
(1)甘藷貯藏蛋白質sporamin合成的調控7
(2)馬鈴薯貯藏蛋白質patatin合成的調控8
(3)芋頭(Colocasia)貯藏蛋白質tarin合成的調控10
(4)山藥貯藏蛋白質dioscorin合成的調控11
第四節、植物生產蛋白質的表現系統11
第五節、基因轉殖植物表現的分析系統13
第二章、Dioscorin基因之基因組 DNA 的選殖15
第一節、前言15
第二節、材料與方法16
第三節、結果與討論20
第三章、Dioscorin基因之基因組序列的分析22
第一節、前言22
第二節、材料與方法22
第三節、結果與討論24
第四章、TAIL-PCR選殖dioscorin基因的側邊序列26
第一節、前言26
第二節、材料與方法27
第三節、結果與討論29
第五章、啟動子序列分析與轉殖載體之構築32
第一節、前言32
第二節、材料與方法33
第三節、結果與討論38
第六章、Dioscorin基因調控區對GUS基因在菸草之暫時性表現分析41
第一節、前言41
第二節、材料與方法41
第三節、結果與討論43
第七章、展望46
參考文獻48
圖表59
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