番石榴ACC合成酶於不同後熟特性番石榴果實基因表現與啟動子分析之研究

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計畫名稱: 番石榴ACC合成酶於不同後熟特性番石榴果實基因表現與啟動子分析之研究
計畫主持人: 吳俊達
共同計畫主持人:
計畫編號: MOST103-2313-B002-008-MY2
計畫主管機構: 科技部
計畫執行機構: 國立臺灣大學園藝暨景觀學系暨研究所
全程計畫年: 2015
關鍵字: 番石榴;後熟;乙烯;ACC 合成酶;啟動子; Guava; Ripeing; Ethylene; ACC synthase;Promote
摘要: 番石榴(Psidium guajava L.)為熱帶美洲原產之桃金孃科(Myrtaceae)經濟果樹, 其栽培種依其果實後熟特性可分為採後易變軟、不耐貯運的更年性品種,如‘梨仔拔’, 和果肉清脆、耐貯運的非更年性品種,如‘珍珠拔’。 ‘珍珠拔’無法後熟並非源於果實 組織對乙烯不敏感,而是其綠熟果組織缺乏 ACC(1-aminocyclopropane-1-carboxylic acid)合成酶(ACS)活性,因而無法提供足量 ACC 供大量乙烯合成所致。系統-2 ACS 基因 PgACS1 為番石榴果實後熟時期乙烯生合成的關鍵酵素基因,雖然存在於‘珍珠拔’ 基因組,卻無法表現,現行假說認為其啟動子缺失所致。本計劃擬將以染色體步移技 術分別自‘梨仔拔’與‘珍珠拔’番石榴基因組選殖 PgACS1 基因啟動子序列進行比較,以 瞭解該基因在兩參試品種調控的差異,並以基因誘導處理與 GUS 報導基因暫時性表現 驗證序列分析結果。另外,根據 PgACS1 啟動子核苷酸序列資訊設計的專一性 PCR 引 子(primer),檢測不同後熟特性番石榴品種葉片抽取基因組 DNA 之 PCR 反應產物, 評估作為鑑別後熟特性 DNA 分子標記之可行性。本計劃結果除了闡釋番石榴品種間不 同後熟性狀的分子機制外,亦可應用於番石榴雜交育種計劃後裔早(苗)期選拔果實 後熟特徵之篩檢 DNA 標記。Guava (Psidium guajava L.), a member of Myrtaceae, is a cash fruit crop native to tropic American. According to the ripening behavior, guava cultivars can be divided into two groups: climacteric fruit-type varieties, e.g. ‘Li-Tzy Bar’, with soft pulp and very short shelflife due to their rapid rate of ripening, and nonclimacteric fruit-type varieties group, e.g. ‘Jen-Ju Bar’, with crispy pulp and better storability. The ripening characteristics of ‘JJB’ did not result from a defect in ethylene sensitivity, but instead result from a lack of ACC (1-aminocyclopropane-1-carboxylic acid) synthase (ACS) activity and, therefore, ACC production for massive ethylene synthesis. PgACS1, a System-2 ACS gene, encoded the key enzyme responsible for the ethylene synthesis during guava ripening. The working hypothesis considers the fact that though PgACS1 did exist in the genome of ‘Jen-Ju Bar’, its expression was barely detectable, is a result of defection in the promoter region of the gene. To reveal the regulatory mechanism, this proposal plan to clone the PgACS1 promoters from ‘Li-Tzy Bar’ and ‘Jen-Ju Bar’, individually, via genomic walking technology and then to compare the differences of the two nucleotide sequences. The bioinformatics results will be verified by induction treatment and transient express of GUS reporter gene system. Furthermore, specific PCR primer pairs designed base on the information of promoter sequence will be utilized in PCR reaction with genomic DNA isolated from leaves of guava with different ripening-behavior characteristics and to evaluate the feasibility as DNA markers for discrimination of ripening behavior in guava. The results of this research will not only to clarify the underline molecular mechanism of different ripening behavior among guava varieties, but also to utilizing as a molecular marker for early (seedling) stage selection among progeny in a breeding program.
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