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研究生:張乃惇
研究生(外文):Chang, Nai-Tun
論文名稱:番石榴植株花青素色素形成的遺傳及R2R3-MYB和花青素生合成基因的特性分析
論文名稱(外文):Inheritance of anthocyanin pigmentation and characterization of R2R3-MYB genes and anthocyanin biosynthetic genes in guava (Psidium guajava L.)
指導教授:陳幼光
指導教授(外文):Chen, Yu-Kuang
口試委員:陳福旗鄭萬興
口試委員(外文):Chen, Fure-ChyiCheng, Wan-Hsing
口試日期:2017-01-19
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:農園生產系所
學門:農業科學學門
學類:一般農業學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:69
中文關鍵詞:番石榴花青素R2R3-MYB 基因後裔檢定
外文關鍵詞:guava (Psidium guajava L.)anthocyaninR2R3-MYB geneprogeny test
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為了解花青素性狀在番石榴的遺傳及表達,比較綠葉綠果皮粉紅肉的番石榴品系V31及其紫紅色自交後代V31(X)-1。V31(X)-1在小果期果肉花青素含量最高,但隨果實發育膨大花青素含量逐漸降低。親本V31在果實發育各個階段則未檢測到花青素。由‘珍珠拔’基因組資料庫中挑選出的93個R2R3 MYBs基因中,有五個番石榴PgMYBs與阿拉伯芥調控花青素生合成的MYBs同屬一亞群,分別命名為PgMYB1 ~ PgMYB5。番石榴PgMYB1與來自其他物種調控花青素生合成的MYBs在R2R3 domain有很高的胺基酸序列相似性。與‘珍珠拔’相比,V31(x)-1在PgMYB1啟動子的GA重複區增加了31次的重複。即時定量PCR分析果肉中五個PgMYBs,發現V31(x)-1的PgMYB1與花青素表達趨勢一致。在花青素生合成早期的基因包括PgCHS、PgCHI、PgF3H和PgDFR的表達也與花青素含量大致相同。然而,V31的PgMYBs和花青素生合成基因的表達與果實的花青素含量並無相關性。V31自交系及其衍生子代的遺傳分析顯示在該族群中花青素色素累積的遺傳由單一基因控制。結果指出綠色等位基因對紫紅色等位基因為顯性,且V31為異質結合。由PgMYB1啟動子區設計的專一性引子所擴增的條帶在28個番石榴基因型中有很高的多型性。該引子對可作為品種鑑定的標記。
In order to understand the genetics and expression of anthocyanin pigmentation in guava, a green-leaf, green-exocarp, and pink-fleshed line V31 and its purple selfed progeny V31(x)-1 were compared. V31(x)-1 had the highest anthocyanin content at small fruit stage, but its anthocyanin content gradually decreased as the fruit developed. Anthocyanin was not detected in V31 at all developmental stages of the fruit. Among 93 R2R3 MYBs genes selected from the ‘Jen-Ju Ba’ genomic database, five guava PgMYBs were grouped into the same cluster as Arabidopsis MYB genes regulating anthocyanin biosynthesis and were named PgMYB1 to PgMYB5. R2R3 domains in guava PgMYB1 and many anthocyanin biosynthesis-controlled MYBs from other fruits exhibit high amino acid similarity. When compared with that of ‘Jen-Ju Ba’, the GA repeat number in the PgMYB1 promoter region of V31(x)-1 is larger by 31 repeats. Among the five PgMYBs, the expression of PgMYB1 in V31(x)-1 fruit flesh by qPCR analysis was consistent with the expression pattern of anthocyanin. The expressions of PgCHS, PgCHI, PgF3H and PgDFR in earlier anthocyanin biosynthesis were also concordant with anthocyanin content in general. However, the expressions of PgMYBs and anthocyanin biosynthetic genes in V31 were not correlated with the anthocyanin content of fruit. Genetic analysis in V31 selfed lines and their derived progeny has revealed that inheritance of anthocyanin pigmentation in this population is controlled by one single gene. The result indicated that the green allele is dominant to the purple allele and V31 is heterozygous. Fragments amplified by the specific primers designed from PgMYB1 promoter region showed high polymorphism in 28 guava genotypes. This primer pair can be used as a marker for cultivar identification.
目錄
頁次
摘要 I
Abstract III
目錄 IV
圖表目錄 VI
壹、前言 1
貳、前人研究 2
一、臺灣番石榴的品種演進與產業現況 2
二、番石榴植物特性 2
(一)番石榴果實性狀 2
(二)果實的營養成分 3
三、花青素 3
(一)花青素的生合成路徑 3
(二)影響花青素表達的因子 4
四、R2R3 MYB 轉錄因子 7
(一) R2R3 MYB 轉錄因子的特徵 7
(二) R2R3 MYB調控果實花青素的表達 9
(三) MYB與其他轉錄因子的交互作用 10
(四) MYB花青素表現性狀的分子標記 11
五、花青素相關性狀的遺傳 12
參、材料與方法 14
一、植物材料 14
二、總量DNA萃取 14
三、總量RNA萃取 15
四、單股cDNA合成 16
五、番石榴PgMYBs及花青素生合成結構基因篩選 16
(一) PCR產物之膠體純化 16
(二)載體接合作用 17
(三)轉型作用 17
(四)菌落藍白篩與菌落PCR 17
(五)菌液增殖 18
(六)小量抽取質體 18
(七)定序及資料判讀 18
六、MYB基因啟動子序列選殖 19
七、雜交授粉及實生苗培育 19
八、定量聚合酶連鎖反應(quantitative PCR, qPCR) 19
九、番石榴R2R3 MYB親緣樹狀圖繪製 20
十、果肉花青素萃取 20
肆、結果
一、V31自交與雜交後代果實顏色的表現 21
二、V31和V31(x)-1果實的發育及花青素含量的變化 21
三、番石榴PgMYB1之特徵 21
四、PgMYB1與其他花青素相關R2R3 MYBs基因之親緣關係 22
五、番石榴花青素結構基因分析 23
六、V31與V31(x)-1在PgMYB1之差異 23
七、PgMYB1 啟動子選殖 23
八、PgMYB1啟動子區域順式表達元件分析 24
九、V31品系不同雜交組合後代花青素性狀之遺傳 24
十、番石榴R2R3 MYBs之qPCR表達分析 25
十一、果肉花青素生合成結構基因之qPCR表達 26
十二、番石榴PgMYB1啟動子專一性引子對於品種鑑定上的應用 26
伍、討論
一、花青素在番石榴及其他物種果實的表達 51
二、番石榴R2R3 MYBs特性 51
三、番石榴花青素性狀遺傳 52
四、番石榴R2R3 MYBs基因之qPCR分析 53
五、番石榴花青素結構基因之qPCR分析 54
六、 鑑別番石榴基因型分子標記的開發 55
陸、結論 56
柒、參考文獻 57
作者簡介 69
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