不同後熟特性番石榴品種ACC氧化酶cDNA之選殖與分析

Cloning and Characterization of 1-Aminocyclopropane-1-carboxylate Oxidase cDNAs from Guava（Pisdium guajava L.） Varieties with Different Ripening-behaviour

劉德駿(Te-Chun Liu)

指導教授 ： 吳俊達

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.02140

摘要

番石榴（Psidium guajava L.）為常綠熱帶果樹，果實富含維生素、礦物質及具抗氧化能力之特點，為臺灣極具發展潛力之經濟果樹。本島栽培番石榴品種依果實後熟特性可分為更年型與非更年型。試驗分析目前臺灣常見的非更年型番石榴栽培品種：‘珍珠拔’、‘水晶拔’、‘帝王拔’、‘二十世紀拔’及大村‘紅心拔’，果實採收後於20℃貯藏204 hr過程之乙烯生成率皆低於 0.20 μL C2H4•kg-1•hr-1，試驗期間皆沒有顯著乙烯生成的變化；相較於更年型品種‘梨仔拔’果實於20℃環境貯藏，其乙烯生成率可達71.02 μL C2H4•kg-1•hr-1，明顯高於非更年之品種。非更年型番石榴品種於自然環境下果實缺少大量的自生乙烯，而無劇烈軟化、轉色、濃郁香味生成的後熟反應，因此具有較長的儲運壽命與商品價值；而另以外施丙烯處理的結果顯示，丙烯可誘導非更年型番石榴果實進行後熟相關的生理變化，卻無法產生大量系統Ⅱ內生乙烯，顯示其後熟機制應與乙烯之生合成能力有著密切的關係，而非乙烯感受能力的差異所致。
以RT-PCR（reverse transcriptase- polymerase chain reaction）策略針對‘珍珠拔’及‘梨仔拔’兩品種番石榴組織進行ACO cDNA之選殖，兩品種果實皆可分離出兩個相同的番石榴cDNA選殖系，分別具有1,259及1,241個核苷酸，推估可轉譯出321個胺基酸，具有ACO活性相關的12個胺基酸與 4 個白胺酸拉鍊（leucine zipper）的結構位，其估算分子量為36.21及36.28 kDa，等電點（pI）為5.39 及 5.78。進一步將兩cDNA選殖系由大腸菌異源表現系統表達，皆能產生具有ACO活性的融合蛋白質，確認其為番石榴 ACO cDNA選殖系，分別命名為Pg-ACO1及Pg-ACO2。南方氏墨點法分析結果推估ACO基因在番石榴組基因的拷貝數約為4至5個。
北方墨點分析及RT-PCR分析Pg-ACO1及Pg-ACO2的表現結果，番石榴果實及枝條具有Pg-ACO1及Pg-ACO2轉錄產物的累積，而花朵及葉片無顯著轉錄產物的累積。Pg-ACO1於‘珍珠拔’與‘梨仔拔’分別於花後第60及120天持續表現至採後第12天，轉錄產物累積於不同天數並無明顯變化；Pg-ACO2於‘珍珠拔’與‘梨仔拔’皆於花後第15天幼果可偵測到轉錄產物的累積，隨後表現降低，分別於花後第60及120天才又恢復mRNA累積的情形。‘梨仔拔’果實Pg-ACO1及Pg-ACO2轉錄產物累積量於後熟過程皆有顯著上升趨勢，‘珍珠拔’則於採後貯藏期間持續表達。以100 μL•L-1乙烯及10 mg•L-1 1-MCP處理‘梨仔拔’番石榴綠熟果實，結果顯示Pg-ACO1及Pg-ACO2皆可受到乙烯誘導而增加轉錄產物的累積量；而1-MCP處理則具有壓抑兩者表達的效果，因而推估兩者皆具“系統Ⅱ”乙烯生成特性的ACO cDNA選殖系，兩選殖系皆可於‘珍珠拔’果實發育第60天即可偵測到轉錄產物的累積，又可於果實後熟階段表達，兩者表現的特性類似番茄無明顯系統區分的ACO基因，皆能參與果實乙烯生成系統Ⅰ與系統Ⅱ期間的乙烯生合成。果實組織酵素活性分析的結果得知，‘珍珠拔’果實組織ACO活性於採後第0~10天皆顯著高於‘梨仔拔’果實組織，顯示兩類型品種果實組織皆具催化ACC為乙烯的能力。
由上述結果指出，‘梨仔拔’與‘珍珠拔’之後熟特性差異並非Pg-ACO1及Pg-ACO2無法表達或缺乏ACO活性所造成。Pg-ACO1及Pg-ACO2於番石榴果實乙烯生成並無明顯的系統區分，Pg-ACO2為果實後熟主要表達的ACO。

關鍵字

番石榴 ； 乙烯 ； ACC氧化酶後熟 ； 更年 ； 選殖

並列摘要

Guava (Psidium guajava L.) is a tropical evergreen fruit crop of Myrtaceae, and its fruit is rich in vitamins and minerals and has antioxidant capacity. The guava cultivars in Taiwan are divided into 2 groups, climacteric and non-climacteric, based on their ripening behavior. Five of the common non-climacteric guava cultivars in Taiwan, ‘Jen-Ju Bar’, ‘Shuei-Jin Bar’, ‘Di-Wang Bar’, ‘Er-Shr-Shr-Ji Bar’ and ‘Hung-Shin Bar’, and one of the climacteric guava cultivar, ‘Li-Tzy Bar’, were used to investigated the postharvest physiology. The ethylene production of non-climacteric fruits remained under 0.20 μL•kg-1•L-1 during 204 hours storage at 20℃, while that of climacteic fruits could reach 71.02 μL•kg-1•L-1. Treatment of exogenous propylene induced the ripening related response of non-climacteric cultivar guava, indicating that the ripening mechanism in cultivars of this group is closely linked with ethylene synthesis instead of ethylene perception.
The two ACO cDNA clones in ‘Jen-Ju Bar’ and in ‘Li-Tzy Bar’ tissues were isolated by RT-PCR (reverse transcriptase- polymerase chain reaction). The numbers of base pairs, molecular weights of the derived amino acid sequence, and the pI value of these two cDNA clones were 1,259 and 1,241, 36.21 and 36.28 kDa, and 5.39 and 5.78, respectively. It was estimated that both of the cDNA clones were able to translate 321 amino acids, in which with 12 ACO activity related conserved residues and 4 leucine zipper sites. Fusion proteins of the two cDNA clones obtained by using E. coli heterologous expression system demonstrated ACO enzymatic activity, which confirm that the two cDNA clones are guava ACO cDNA clones, and thus named as Pg-ACO1and Pg-ACO2.The result of Southern blotting suggested that the copy number of ACO genes in guava genome is approximately 4 or 5.
The results of northern blotting and RT-PCR assay on Pg-ACO1and Pg-ACO2 showed that there was accumulation of transcripts of Pg-ACO1and Pg-ACO2 in guava fruits and shoots but not significant in flowers and leaves. Pg-ACO1 in ‘Jen-Ju Bar’ and in ‘Li-Tzy Bar’ constitutively expressed from 60 and 120 days after anthesis, respectively, to 12 days during postharvest. Pg-ACO2 transcripts in ‘Jen-Ju Bar’ and in ‘Li-Tzy Bar’ were detectable in the 15th day after anthesis, and then decreased until the 60th and 120th day after anthesis, respectively. Transcripts of Pg-ACO1and Pg-ACO2 in ‘Li-Tzy Bar’ fruit significantly increased during ripening, and that in ‘Jen-Ju Bar’ constitutively expressed during storage. The results of 100 μL•L-1 ethylene and 10 mg•L-1 1-MCP treatments showed that the transcripts of Pg-ACO1 and Pg-ACO2 in mature green ‘Li-Tzy Bar’ were both increased by ethylene elicitation and suppressed by 1-MCP. Therefore, it was inferred that two ACO cDNA clones possess the characteristics of “system Ⅱ” ethylene synthesis. However, in ‘Jen-Ju Bar’ fruits, the two ACO transcripts were detected from 60th day after anthesis to 12th day postharvest suggesting that both ACO clones might be involved in ethylene biosynthesis in both fruit development and ripening stage and that the two ACO transcripts possess characteristics of both “system Ⅰ” and “system Ⅱ”. In addition, the ACO activity of ‘Jen-Ju Bar’ was significantly higher than that of ‘Li-Tzy Bar’ during the first 10 days postharvest.
The results shown above indicate that the difference in the ripening behaviors between ‘Li-Tzy Bar’ and ‘Jen-Ju Bar’ is neither caused by silence of Pg-ACO1and Pg-ACO2 nor by the lack of ACO activity. Furthermore, according the ethylene and 1-MCP experiment on ACO transcripts in ‘Jen-Ju Bar’ fruits, it is hard to conclude which system of ethylene synthesis that both of Pg-ACO1 and Pg-ACO2 belong to.

並列關鍵字

guava ； ethylene ； ACC oxidase ； ripening ； climacteric ； cloning

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