Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36911
標題: 大豆種皮異類黃酮生合成基因IFS及IFR之選殖與轉殖大豆調節基因之菸草花色素組成分變化
Molecular Cloning of IFS and IFR in Seed Coats of Soybean (Glycine max) and Alteration of Pigment Compositions in the Flowers of Transgenic Tobacco (Nicotinan tabacum L.) transformed with Soybean Regulatory Gene.
作者: 梁心怡
Liang, Shin-Yi
關鍵字: Glycine max;isoflavone synthase;isoflavone reductase;regulatory gene;pigment
出版社: 農藝學系
摘要: 
異黃酮 (isoflavones) 為一種酚類化合物,其化學結構與功能與動物雌激素很類似,亦稱作植物雌激素 (phytoestrogen) ,且為生合成植物防禦素 (phytoalexins) 之前驅物。IFS (isoflavone synthase) 及IFR (isoflavone reductase) 基因為生合成異黃酮之關鍵酵素,本論文目的為選殖大豆 (Glycine max) Clark 近同源品系 (near isogenic lines) L66-14 (UC9) 之IFS及IFR基因。根據根據已發表大豆之IFS (AF195798和AF195799) 及IFR1 (AF202183) 核酸序列設計引子 (primers) ,進行逆轉聚合酶鏈鎖反應,得到含預期片段之IFS12-25及IFR1-5殖系,經由SDSC網站 (http://workbench.sdsc.edu/)針對其核苷酸及胺基酸序列進行比對,確認得到IFS基因及IFR1 cDNA全長。針對不同基因型之大豆品系UC1 (IRT) 、UC9 (iRT) 及UC30 (iRt) ,經由南方轉漬分析 (Southern blot analysis) 顯示,IFS基因和IFR1基因與控制花青素之基因座I、R及T沒有關係。
本論文另一試驗為進行轉殖大豆myc及myb之菸草植株子代之篩選,與分析其花瓣花青素含量之變化。本研究將由大豆所選殖出之myb及myc基因,接上CaMV35S promoter和NOS terminator,分別構築於載體pCAMBIA1300及pBI121,再利用農桿菌系LBA4404進行轉殖。比較轉殖myc基因和轉殖myb與myc兩個基因之轉殖菸草植株,其花青素種類及含量之差異。以HPLC分析T1世代植株花瓣花青素種類及含量,發現總花青素含量皆明顯增加,更進一步細看其所含花青素種類,發現除了cyanindin之外,部分植株更有pelargonidin之累積;比較單獨轉殖myc基因子代之cyanindin含量約為wild type之210 %;而轉殖myb及myc二基因之子代cyanindin含量並未大量增加,但其pelargonidin含量約為同樣含有pelargonidin之轉殖myc基因菸草的230 %。綜合南方雜交分析與HPLC的結果可知,僅轉殖myc基因會造成cyanidin大量合成,而使花色變為深紅色;同時轉殖myb及myc兩個基因,會造成pelargonidin大量生合成,而使花色偏向磚紅色,而基因插入套數多寡與花青素的含量並無絶對的關係,欲証明轉殖轉錄調節基因myc與myb對於花色的影響,仍需藉由北方轉漬分析花青素生合成構造基因之表現量才可得知。

Isoflavone is a kind of phenolic compounds, with chemical structure and function similar estrogen, also known as phytoestrogen. Isoflavone is the precursor to the phytoalexins. IFS (isoflavone synthase) and IFR (isoflavone reductase) are key enzymes to synthesize isoflavones. One of the purpose of this thesis is to clone the cDNA encoding IFS and IFR1 by RT-PCR from soybean seed coat to study their relationship between Clark near isogenic lines. Two complete cDNAs, IFS12-25 (1,702 bp) and IFR1-5 (924 bp), coding for IFS and IFR1 were cloned from seed coats of the black soybean variety UC9, respectively. The deduced amino acid sequences of IFS12-25 clone is with 89 % identity to Glycine max P450 monooxygenase protein by BLAST analysis of NCBI. And the IFR1-5 clone is with 99 % to Glycine max IFR1 protein. Southern blot analysis indicated that IFS and IFR1 gene are not related with I, R, T loci , which controlling anthocyanin synthesis, and will not effect isoflavones synthesis directly.
The other purpose of this thesis is to screen transgenic tobacco T1 plants, and analyze their petal anthocyanin content. Two anthocyanin regulatory genes, myc and myb , of soybean were introduced into tobacco in a strategy designed to stimulate the anthocyanin pathway and alter the composition of anthocyanin produced in flower. Myc and myb gene constructs included full-length gene, double CaMV 35S promoter and NOS terminator. HPLC analysis indicated tobacco petals, which over expression myc gene, with higher 2.1 fold cyanidin as of wild type. Over expression of both myc and myb genes tobacco petals contain higher total anthocyanins, with normal cyanidin and higher pelorgonidin. The result support regulator genes are of potential to enhance expression of anthocyanin pathway structure genes, and increase anthocyanin synthesis or change the direction of anthocyanin pathway and influence the flower color.
URI: http://hdl.handle.net/11455/36911
Appears in Collections:農藝學系

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