臺灣番石榴/番荔枝之親緣關係與果實後熟相關基因之研究

Abstract

番石榴 (Psidium guajava L.) 與番荔枝 (Annona squamosa) 為台灣重要的果樹，除一般作鮮食外，尚具有高加工和傳統醫學的利用性。台灣野生芭樂之中，紅心芭樂在民俗醫學上最具價值，常見於台灣原住民部落。但由於紅心芭樂並無大量栽種，年產量不定，常有以其他芭樂摻假的情況發生。本研究利用遺傳分子鑑定技術，蒐集台灣原住民各部落之土芭樂進行遺傳物質分析，以建立摻假之辨識系統，並了解各品種土芭樂在原住民部落分佈之情形。番荔枝果實在台灣因改良品種繁多，以果實之外觀形態作為品種區分的依據有者亦不易區分，本研究以分子標誌技術，建立快速鑑別各品種番荔枝之方法，加速果實品種的改良和優良性狀之篩選。 本研究第一部份分別針對土芭樂及番荔枝核糖體DNA之18S與ITS1、葉綠體DNA之trnL intron與trnL-trnF IGS等區域進行分析，並以NJ、PA、ML及UPGMA演算法計算各區域之演化樹狀圖。結果發現，以18S rRNA可明確的將各部落之土芭樂依地緣之相近性作區分，在各演算結果均一致；以RAPD方法分析，不但可區分紅、白心芭樂，亦可將經濟品種番石榴與土芭樂作區隔，以及不同海拔與緯度環境之土芭樂作區分。在番荔枝的親緣關係分析方面，葉綠體DNA之trnL-trnF IGS序列分析、演算法計算所得之結果，均印證過去以形態作為分類的基礎，可節省果樹生長的時間，適合作為不同品種番荔枝親緣關係分析之鑑定方法。 第二部份的研究則是探討兩種果樹之果實軟化相關之聚半乳糖醛酶 (polygalacturonase, PG)、乙烯合成關鍵酵素ACC合成酶 (ACC synthase, acs) 與ACC氧化酶 (ACC oxidase, aco) 等後熟基因。紅心芭樂的PG基因 (P. guajava PG, PgPG) 與鳳梨釋迦的PG基因 (Atemoya PG, AtPG) 部分序列之大小分別為346 與351 bp，所推衍之胺基酸序列分別為115及116個胺基酸；此兩段胺基酸序列與其他物種之PG進行排列分析，PgPG與AtPG含有不完整之open reading frame (ORF) 以及有四個高度保守性區域，含有和前人相同之三個胺基酸Gly-His-Gly (GHG) 與保守之Tyr殘基，為PG催化活性部位，具有endo-PG之活性。紅心芭樂ACC氧化酶基因 (P. guajava ACC oxidase gene, PgACO)之部分序列長度為729 bp，推衍出242 bp個胺基酸，經排列比對、結構分析與ORF預測等，PgACO之胺基酸序列有七個保守性的區域，其中高保守性的七個胺基酸殘基為dioxygenase的Fe (II) ascorbate family；後熟過程PgACO因受到乙烯的誘導而表現。鳳梨釋迦ACC合成酶基因 (Atmoya ACC synthase gene, AtACS) 之部分序列長度為990 bp，推衍出323個胺基酸，根據序列經排列比對、結構預測等結果，具有七個高度保守性區域，序列中八個保守胺基酸與aminotransferase有關，且含有決定ACC synthase活性之TNP domain，故證實本研究之AtACS具備ACC synthase的活性。 第三部分使用real-time PCR分析兩種果樹後熟基因之表現。紅心芭樂之PgPG受到乙烯的誘導，在後熟第三天PG酵素活性增加，果實軟化速度加快；PgACO 在果實未後熟時有些微的表現，經乙烯誘導第二至四天的表現量最高，此時紅心芭樂果實之顏色由綠轉黃，並持續表現至後熟末期。鳳梨釋迦之AtPG基因的表現與果實軟化速率相關，後熟第二天AtPG有最高表現量，此階段鳳梨釋迦果肉軟化速率最高。而由AtACS的表現得知ACC synthase活性增加，乙烯在整個後熟期間持續生成。本研究所建立之分子遺傳鑑別系統和果實軟化與乙烯合成之關鍵的基因對栽培、採收後和加工的利用上都將有實質更進一步開發和應用潛力。

Guava and sugar apple are important fruit trees in Taiwan. Red-flesh guava is mostly distributed in indigenous tribes in Taiwan and has valuable application in traditional medicine. Due to the various varieties of red-flesh guava and adulteration problems in fruit materials, a systematic way for classification using molecular marker is necessary to guarantee materials’ reliability and safety. Same manner in sugar apple, there are numerous cultivars and species due to breeding for fruit improvement, thus, reliable molecular detection method is urged for rapidly screening and maintenance of species quality. In the first part, ribosomal DNA 18S, ITS1 and chloroplast DNA trnL intron and trnL-trnF IGS were used for the identification of guava and sugar apple samples. The results were calculated by algorithm NJ, PA, ML and UPGMA. The results showed similarity in each DNA regions. Analysis of uncultivated guavas’ rDNA 18S marker revealed a correlation with their geographical distribution in tribes. In addition, molecular marker of indigenous guava was found different from commercial cultivars, it also reflect differences on altitude and latitude, and red and white-flesh guava. In term of sugar apple, the results showed that cpDNA trnL-trnF IGS was agreeable to morphological classification and suitable for identifying different species and cultivars sugar apples. In the second part, the ripening gene of red-flesh guava and atemoya were identified and characterized. The partial cell wall degradation related genes, PgPG of guava and AtPG of atemoya, were 346 and 351 bp and the deduced amino acid sequences were 115 and 116 in length, respectively. The PgPG and AtPG deduced amino acid aligned with others have an uncompleted OFR and four highly conserved regions that with catalytic sites Gly-His-Gly (GHG) and Tyr residue. On the other hand, PgACO, a ACC oxidase gene from red-flesh guava was 729 bp with 242 deduced amino acids. The alignment of PgACO showed seven conserved regions. There are 9 conserved amino acid residues belongs to dioxygenase, Fe (II) ascorbate family. The expression of PgACO was induced by ethylene. ACC synthase gene (AtACS) of atemoya was 990 bp with a 323 deduced amino acids. Eight amino acid residues in seven conserved regions were correlated with aminotransferase. TNP domain of conserved region was a key site for activity of ACC synthase. In the third part, the ripening-related gene expression of red-flesh guava and atemoya were studied by real-time PCR. PgPG and PgACO were induced by ethylene and firmness loss rate of fruit increased while PG activity was high. The skin color of Red-flesh guava turned yellow as high transcription level of PgACO was detected during ripening from 2nd day to 4th day. AtPG was correlated with the rate of fruit soften, and AtACS induced ACC synthase for ethylene production during fruit ripening. The established molecular detection system, and identified ripening-related gene of guava and sugar apple would be useful for further application in cultivation, postharvest and processing.