番石榴果實對乙烯敏感性與其受體cDNA選殖表現分析

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論文名稱: 番石榴果實對乙烯敏感性與其受體cDNA選殖表現分析
研究生姓名: 葉孋嬋
指導教授姓名: 吳俊達
出版年: 2013
學校名稱: 國立臺灣大學
系所名稱: 園藝暨景觀學系
關鍵字: 乙烯;丙烯;番石榴;乙烯受體;敏感性;ethylene;propylene;guava;ethylene receptor;sensitivity
摘要: 非更年型ʻ珍珠拔ʼ番石榴果實自身不會產生系統二乙烯之特性,可排除內生乙烯影響,適合作為探討番石榴果實後熟各項生理反應對乙烯敏感性的材料。番石榴果實軟化與轉色對丙烯(乙烯類似物)具高度敏感性(分別為11.94與14.26 μL.L-1丙烯)、反應性(2.25 N/μL.L-1丙烯與1.67°/μL.L-1丙烯)及依賴性(91.9%與95.9%),所以ʻ珍珠拔ʼ於20℃暴露於1000 μL.L-1丙烯4 h就足以明顯影響果實硬度,處理時間延長至24 h果皮色相角顯著下降。然而果實總可溶性固型物與可滴定酸含量在後熟過程則對丙烯的依賴性低,僅為28.9%與20.2%,暗示果實採收成熟度才是主要影響因子。乙烯訊號需藉由乙烯受體被植物感知,由ʻ珍珠拔ʼ及ʻ梨仔拔ʼ番石榴果實組織分離出番石榴乙烯受體PgETR1及PgETR3 cDNA選殖系。PgETR1全長3027 bp.,由核苷酸序列推估可轉譯733個胺基酸;PgETR3片段長度則為542 bp.。PgETR1及PgETR3皆受乙烯誘導表現量上升,兩者在果實發育期間表現趨勢類似,花後5-30天表現高峰可能與幼果期大量乙烯生成誘導有關;花後45-75天則推測為生長素濃度提高所致。ʻ珍珠拔’果實採後並無明顯乙烯生成,因此PgETR1及PgETR3在採後基因表達量並無顯著變化;而ʻ梨仔拔’果實後熟期間PgETR1與PgETR3具有不同的表現趨勢,PgETR1於果實後熟期間表現量無增加趨勢,而PgETR3則隨果實後熟天數增加而逐漸上升,顯示PgETR3可能調控ʻ梨仔拔’果實後熟時對乙烯的敏感性。分析不同濃度丙烯處理24 hʻ珍珠拔ʼ果實其PgETR1及PgETR3表現,顯示兩乙烯受體表現量隨丙烯濃度增加而上升。而ʻ珍珠拔ʼ不同時間1000 μL.L-1丙烯處理果實其PgETR1及PgETR3在處理4 h後顯著增加,且4-72 h維持穩定的表現,對照‘珍珠拔’果實經4 h丙烯處理後放置3天果實硬度顯著下降,推測4 h丙烯接觸足以使果實乙烯受體不活化,無法抑制乙烯訊息傳遞,開啟乙烯調控基因表現,而誘發果實後熟生理反應。‘Jen-Ju Bar’ (‘JJB’) guava, a non-climecteric cultivar which defects in system II ethylene production during fruit ripening stage, is an appropriate material for exploring ripening responses to ethylene sensitivity of guava fruit, because the impacts of endogenous ethylene can be excluded. The pulp softening and peel coloration of ‘JJB’ fruit showed a high sensitivity (11.94 and 14.26 μL.L-1 propylene, respectively.), responsivity (2.25 N/μL.L-1 propylene and 1.67°/μL.L-1 propylene) and dependency (91.9% and 95.9%) for propylene, an ethylene analogue. As a consequence, the minimal durations affecting firmness and peel color of exposure in 1000 μL.L-1 propylene at 20℃ were 4 and 24 h, respectively. In contrast, during ripening, the changes of total soluble solids and titratable acidity of ‘JJB’ exhibited a low propylene dependency manner, only 28.9% and 20.2%, which implys that harvest maturity is the major influence factor for both attributes in ‘JJB’. Since the perception of ethylene occurs when ethylene binds to its receptor in planta, two cDNA clones, namely PgETR1 and PgETR3, of ethylene receptors were isolated from the pulp tissues of ‘JJB’ and ‘Li-TzyBar’ (‘LTB’), a climacteric guava cultivar. The numbers of nucleotide and derived amino acid residues of PgETR1 were 3027 and 733, respectively. PgETR3, a partial cDNA clone, has 542 nucleotids. The two genes were up-regulated by exogenous ethylene and showed nontissue-specific expression patterns. During fruit development, PgETR1 and PgETR3 expression patterns were similar, expression peak of 5-30 days after anthesis may be associated with large ethylene production of young fruit; expression peak of 45-75 days after anthesis was supposed the auxin concentration raise. ‘JJB’ did not produce obviously ethylene during ripening stage, thus PgETR1 and PgETR3 expression remained during postharvest. However, PgETR1 and PgETR3 expression patterns were diferent in ‘LLB’ during fruit ripening. PgETR1 expression did not countinously increase, but PgETR3 expression increase gradually, which indicated PgETR3 regulated ‘LLB’ ethylene sensitivity during fruit ripening. ‘JJB’fruit were treated with propylene for different concentration, and analysis PgETR1 and PgETR3 expression was performed, both of them increased with the concentration. PgETR1 and PgETR3 expression raised in ‘JJB’fruit treated with propylene for 4 h, and expressed steadily during 4-72 h treatment. Compared to ‘JJB’ fruit firmness lost, suggested that ‘JJB’fruit suffered propylene for 4 h was sufficient to inactivate ethylene receptors, thus can not suppress ethylene signal transduction, ethylene-related gene were active, and induced fruit ripening responses.
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