生物資源暨農學院: 農藝學研究所指導教授: 常玉強楊琇淳Yang, Hsiu-ChunHsiu-ChunYang2017-03-062018-07-112017-03-062018-07-112015http://ntur.lib.ntu.edu.tw//handle/246246/276066轉位子釣取系統 (transposon tagging) 是利用轉位子 (transposable elements) 創造插入型突變 (insertional mutation) 藉以研究基因功能的技術之一。此系統最大的潛力在於轉位子於生殖細胞轉位 (germinal transposition) 相較於體細胞轉位 (somatic transposition) 可產生較多獨立的插入型突變體。先前本實驗室使用來自玉米Ac / Ds (Activator-Dissociator) 轉位系統，利用水楊酸誘導啟動子PR-1a (Pathogenesis related-1a) 控制Ac轉位酶表現，並將轉位子構築於轉位酶內隱子 (intron)，建構了水楊酸誘導單次轉位系統 (COKC)。而本研究誘導COKC之轉殖水稻生殖細胞轉位，進而配合花藥培養，於單一世代生產同質結合突變體 (homozygous mutants)。該系統分別處理以下兩種水楊酸誘導方式：一、先誘導再分離花藥進行培養。二、同時誘導及花藥培養。前者轉位效率為5%，且皆為同質結合突變體；後者效率為20%，但之中90%為異質結合突變體 (heterozygous mutants)，推測轉位子於花藥培養誘導生殖細胞胚體化 (embryogenesis) 的同時，發生體細胞轉位。為了改善生殖細胞轉位效率，本研究利用水稻花粉不同發育時期之專一表現啟動子 (pollen-specific promoters) OsP1、OsP41和OsP128，建構生殖細胞轉位系統OsP1-0380PL、OsP41-0380PL和OsP128-0380PL，進行水稻及菸草轉殖而後同樣進行花藥培養。綜合上述研究成果，藉由水楊酸誘導系統COKC及生殖細胞轉位系統0380PL，可提升生殖細胞轉位效率，並於單一世代得到穩定的同質結合突變體。Transposon tagging is an effective tool of insertional mutagenesis for studying gene function. A higher number of independent insertion mutants can be obtained from a few transgenic launch pads via germinal transposition. Our lab previously developed a one-time inducible Ac/Ds transposable system, COKC, where the Ds element in the intron of the Ac transposase is driven by the salicylic acid inducible promoter PR-1a (Pathogenesis related-1a). In order to increase germinal transposition and generate homozygous mutants, we treated floral tissues at uninucleate stages of microspore in COKC transgenic rice with salicylic acid in two salicilylic acid treatments. The first approach was pot induction, where rice panicle was induced in the pot prior to anther culture. The other approach was culture induction, where the rice anther was isolated first and then simultaneously induced and incubated. The transposition efficiency of anther-derived regeneration shoots from the two sets were 5 % and 20 %, respectively. For pot induction, all of the regeneration calli were homozygotes. In contrast, for culture induction, most of the regeneration calli were heterozygotes, which may result from somatic transposition after embryogenesis of the callus. In order to increase germinal transposition, the present study aims to construct three novel germinal transposon tagging systems OsP1-0380PL, OsP41-0380PL and OsP128-0380PL. In these systems, the Ac transposase is driven by each of the three pollen specific promoters identified in rice cultivar TNG67. The above tagging constructs were transformed into rice and tobacco through Agrobacterium. After that, the transformants were subjected to anther culture to observe the frequency of transposition in gametophytic process, where more homozygotes could be derived. In conclusion, the aim of this thesis is appling to improve germinal transposition to create stable homozygous mutants in a single generation in plants.3671324 bytesapplication/pdf論文公開時間: 2019/9/17論文使用權限: 同意有償授權(權利金給回饋學校)轉位子釣取系統插入型突變生殖細胞轉位體細胞轉位Ac/Ds轉位子PR-1a啟動子花藥培養同質結合突變體異質結合突變體花粉專一表現啟動子anther culturegerminal transpositiongerminal transposon tagging systemshomozygous mutantsone-time inducible Ac/Ds transposable systempollen-specific promoterspathogenesis related-1asomatic transpositionthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/276066/1/ntu-104-D99621101-1.pdf