不同後熟特性番石榴品種ACC合成酶cDNA選殖與分析

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論文名稱: 不同後熟特性番石榴品種ACC合成酶cDNA選殖與分析
研究生姓名: 陳國恩
指導教授姓名: 吳俊達
出版年: 2009
學校名稱: 國立臺灣大學
系所名稱: 園藝學研究所
關鍵字: 番石榴;ACC合成酶;乙烯;後熟;guava;ACC synthase;ethylene;ripening
摘要: 番石榴(Psidium guajava L.)為桃金孃科(Myrtaceae)之常綠熱帶果樹,原產於墨西哥及祕魯熱帶美洲地區,植株生長強健、風土適應性佳,廣泛栽培於熱帶與亞熱帶地區,也是我國重要經濟果樹之一。番石榴不同品種間,依其果實後熟特性不同可分為更年型與非更年型兩大類。果實後熟特性的差異不僅影響番石榴採後處理與利用,並改變了台灣番石榴產業的發展。而這兩群番石榴品種果實後熟特性的差異原因仍未闡明,因此本試驗的目的在釐清不同後熟特性番石榴品種果實生理與分子層次差異的機制。 ‘梨仔拔’與‘大蒂’番石榴果實採收後呼吸與乙烯生成速率皆有顯著上升趨勢,在20℃環境‘梨仔拔’呼吸速率可達95 mL CO2/kg/hr,‘大蒂’為76 mL CO2/kg/hr;乙烯生成速率最高值分別為33 μL C2H4/kg/hr與16.9 μL C2H4/kg/hr,兩者皆為典型更年型果實。相反的,‘珍珠拔’果實採收後呼吸速率平穩,維持在18∼21 mL CO2/kg/hr之間,而幾乎無乙烯生成,呈現非更年型果實後熟特徵。 ‘珍珠拔’果實經外施丙烯處理可誘導其呼吸速率上升、果實轉黃、硬度下降等乙烯後熟生理進行,但依舊無法促使其果實內生乙烯大量生成。顯示 ‘珍珠拔’果實呈現非更年型後熟特徵乃因果實採收後無法啟動〝系統Ⅱ〞乙烯生合成代謝,並非對乙烯感受或訊息傳導有所缺失。 為更進一步釐清不同後熟特性之番石榴果實在乙烯生合成路徑上之關鍵酵素ACC合成酶(ACC synthase;ACS)基因於轉錄層次調控之差異,針對番石榴組織進行ACS RT-PCR ( reverse transcriptase polymerase chain reaction )基因選殖,獲得兩個番石榴cDNA全長選殖系,長度分別為1830 bp與1861 bp,解碼出491和486個胺基酸,具有典型ACS基因7個保守區跟11個變異性低的胺基酸,極可能為番石榴ACS cDNA選殖系,命名為Pg-ACS1與Pg-ACS2。兩者均屬胺基酸序列C端具有四個磷酸化為之Type Ⅰ ACS。南方氏雜合分析的結果指出Pg-ACS1在‘梨仔拔’與‘珍珠拔’番石榴果實基因組皆為單一拷貝基因;Pg-ACS2為雙拷貝基因。利用JAde 6大腸桿菌表達系統證實番石榴Pg-ACS1與Pg-ACS2選殖系之轉譯產物皆具有ACS活性。 ‘梨仔拔’與 ‘珍珠拔’番石榴果實生長發育曲線皆為雙S型曲線,主要生長為第一及第三階段。果實生長發育至採收階段,果實呼吸速率與乙烯生成皆呈現逐漸下降的趨勢,兩個番石榴品種花後5天的幼果呼吸及乙烯生成速率皆最高,‘梨仔拔’幼果呼吸速率195 mL CO2/kg/hr,‘珍珠拔’為229 CO2/kg/hr; ‘梨仔拔’與 ‘珍珠拔’幼果乙烯產生率分別為0.64 μL C2H4/kg/hr與0.29 μL C2H4/kg/hr。之後呼吸率及乙烯合成速率逐漸降低,直到後熟果階段,‘梨仔拔’呼吸及乙烯合成速率才逐漸上升,進入更年期。Pg-ACS1為負責更年型‘梨仔拔’果實後熟階段大量乙烯生成時期的〝系統Ⅱ〞ACS;受到乙烯的自動催化,綠熟 ‘梨仔拔’果實外施乙烯可誘導其表現,而乙烯作用抑制劑1-MCP處理則壓抑其mRNA累積。Pg-ACS2在 ‘梨仔拔’綠熟前的生長發育階段與非更年型‘珍珠拔’果實整個生長發育過程持續微量表現,其表現也不受乙烯或1-MCP處理的影響,應為一〝系統Ⅰ〞ACS或持續性表現ACS。所以,更年型‘梨仔拔’與非更年型‘珍珠拔’番石榴果實其後熟特性之差異為‘珍珠拔’果實採收後無法啟動〝系統Ⅱ〞ACS基因Pg-ACS1,因此在果實採收後無法合成足夠量ACC,提供後熟階段大量乙烯生成,因而呈現出無法果實轉色、軟化之非更年型果實特徵。Guava (Psidium guajava L.), a tropical evergreen fruit crop of Myrtaceae, is native to in tropical regions of Mexico and Peru. Because of its great environmental adaptability and vigor characteristics, guava is widespread cultivated in tropical and subtropical areas all over the world, and is also a cash fruit tree in Taiwan. According to fruit ripening behavior, guava varieties are generally classified into two groups, namely climacteric and non-climacteric type. The genetic trail of ripening behavior affects not only the way of postharvest handling and utilization of guava, but also the expansion of industrialized cultivation area in Taiwan. Unfortunately, the mechanism(s) causing the different ripening behaviors among guava varieties is still unknown. Therefore, theobjectives of this thesis are to assess the physiological differences in ripening between climacteric and nonclimacteric guava fruits, as well as to investigate the mechanism(s) rendering various ripening behaviors in guava at molecular level. The respiration and ethylene production rate of ‘Li-Tzy Bar’ and ‘Dar-Dih’ guava fruits are significantly increased after harvested. The maximum respiration rate of ‘Li-Tzy Bar’ and ‘Dar-Dih’ at 20 were 95 and 76 mL CO2/kg/hr, respectively; and the peak of ethylene release of these two cultivars were 33 and 16.9 μL C2H4/kg/hr, respectively. The fruits from both cultivars showed the typical characteristics found in climacteric fruits. ‘Jen-Ju Bar’ guava fruit, in contrast, performed as a nonclimateric fruit, since the respiration rate was steady and fluctuated within the range of 18~21 mL CO2/kg/hr as well as the ethylene production was barely detectable in the same storage condition. Although enhancement of respiration, yellowing in peel color, and softening in fruit texture, some typical physiological responses in climacteric fruit ripening, were obviously observed, there was no significant increase of ethylene release from ‘Jen-Ju Bar’ guava fruits after 100 μL/L propylene treatment for 72 hours. These results implies that the reason that ‘Jen-Ju Bar’ guava fruit displayed a nonclimacteric fruit was due to failure of triggering endogenous ‘System Ⅱ’ ethylene synthesis, but not because of defect of ethylene perception and/or signal transduction. Several lines of evidences have demonstrated that ACC (1-aminocyclopropane-1-carboxylic acid) synthase (ACS) is the key enzyme of ethylene biosynthetic pathway in higher plants. In order to illustrate the difference(s) between climacteric and nonclimacteric type guava at molecular level, two full length ACS cDNA clones, 1830 bp and 1861 bp encoding 491 and 486 amino acids, respectively, were isolated from guava tissues by RT-PCR (reverse transcriptase polymerase chain reaction) strategy and used for the further experiments below. Nucleotide sequencing data revealed that there were 7 conserved regions and 11 unvaried amino acids typically found in ACS genes existed in both cDNA clones. They, therefore, were named Pg-ACS1 and Pg-ACS2, respectively. Both genes were considered as Type I ACS genes since four phosphorylated serine sites were found at the C terminal of their derived amino acid sequence. Results of Southern blot analysis showed that Pg-ACS1 was a single copy gene, and Pg-ACS2 was either a single or two copy gene in ‘Li-Tzy Bar’ or ‘Jen-Ju Bar’ guava genome. Moreover, the fact that gene products of Pg-ACS1 and Pg-ACS2 posed ACS enzyme activity was demonstrated by using JAde 6 Escherichia coli expression system. The growth pattern of ‘Li-Tzy Bar’ or ‘Jen-Ju Bar’ guava fruits was a double sigmoid curve characterized with major fruit enlargement in the first and third phase. The highest respiration and ethylene production rate of developing ‘Li-Tzy Bar’ and ‘Jen-Ju Bar’ guava fruit were detected at 5 days after anthesis; and then both gradually declined with fruit development before harvest. The maximum values of respiration rate in developing ‘Li-Tzy Bar’ and ‘Jen-Ju Bar’ guava fruit were 195 and 229 mL CO2/kg/hr; and the maximum ethylene production rate of these two cultivars were 0.64 and 0.29 μL C2H4/kg/hr, respectively. In case of ‘Li-Tzy Bar’ guava, the respiration and ethylene production rate elevated again afterward when the fruit entered ripening stage. Pg-ACS1 expression was attributed to the ACS catalyzed the ethylene production of ‘ethylene burst’ in ripening ‘Li-Tzy Bar’ guava, a climacteric cultivar, based on northern blot analysis. The fact that ethylene treatment induced the gene expression, and 1-methylcyclopropene (1-MCP), an ethylene action inhibitor, suppressed the transcript accumulation in green-mature ‘Li-Tzy Bar’ guava fruits implied the ethylene autocatalysis property of Pg-ACS1 expression, a ‘System II’ ACS. The mRNA of Pg-ACS2 was detected by RT-PCR in developing ‘Li-Tyz Bar’ guava prior to green mature stage and all the stages in ‘Jen-Ju Bar’ guava fruit. Opposite to Pg-ACS1, the expression of Pg-ACS2 was influenced neither ethylene nor 1-MCP in ‘Li-Tyz Bar’ guava. Thus, Pg-ACS2 was considered as a ‘System I’ or a constitutive ACS in guava. In conclusion, the major difference between climacteric ‘Li-Tzy Bar’ and nonclimacteric ‘Jen-Ju Bar’ guava fruit is that the latter variety cannot switch on Pg-ACS1, a ‘System II’ ACS, during ripening. Therefore, no enough ACC to support massive ethylene production in green mature ‘Jen-Ju Bar’ guava fruit and the fruit exhibit nonclimacteric characteristics, including delay yellowing in peel and fruit firmness last longer.
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