請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/83221
標題: | 不同後熟特性番石榴品種關鍵系統-2 ACC合成酶基因PgACS1之基因型分析 Characterization of the Genotypes of PgACS1, a Key System-2 ACC Synthase Gene in Guava, from Cultivars with Different Ripening Behaviors |
其他標題: | Characterization of the Genotypes of PgACS1, a Key System-2 ACC Synthase Gene in Guava, from Cultivars with Different Ripening Behaviors |
作者: | 劉天珠 Margo Sulistio |
指導教授: | 吳俊達 博士 Chun-Ta Wu |
關鍵字: | 番石榴(Psidium guajava L.),後熟,乙烯生合成,ACC合成酶,PgACS1,反轉錄轉位子, Guava (Psidium guajava L.),Fruit ripening,Ethylene biosynthesis,ACC synthase,PgACS1,Retrotransposon, |
出版年 : | 2022 |
學位: | 博士 |
摘要: | 番石榴(Psidium guajava L.)種內具有不同後熟行為之品種,呈現迥異的採後貯運壽命,為研究果實後熟的良好材料;其後熟表現型差異源自果實系統-2乙烯生合成能力不同,然而其分子機制仍未有詳細研究。PgACS1 (JQ280301)為負責番石榴果實後熟大量乙烯生合成的關鍵系統-2 1-aminocyclopropane-1-carboxylate (ACC)合成酶(ACS)基因,本研究探討不同後熟行為番石榴品種PgACS1基因型之差異性,以闡釋品種間乙烯生成量和採後壽命的機制。非更年型‘珍珠拔’(‘JJB’)之PgACS1啟動子-6位置有5,487 bp [含有gag、pol、integrase (IN)、反轉錄酶(RT)、RNase H (RH) 保守基因]的Ty1/Copia長端重複(LTR)反轉錄轉位子Psidium guajava retrotransposon 1 (PgRE1) (LC699797)以反義轉錄方向嵌入,造成Ethylene Response Element (ERE)順式調控元件由-146 ~ -139向上游位移至-5599 ~ -5606,遠離轉錄起始點(Transcription Start Site;TSS),使得該基因無法在果實綠熟階段啟動,因此無法大量合成ACC與乙烯(20℃合成率< 0.1 µL C2H4 kg−1 h−1),果實呈現非更年性表現型,具有較長的採後壽命。此啟動子對於外源丙烯處理呈現不敏感、無反應、不依賴,喪失系統-2 ACS基因乙烯自動催化能力;此基因型被命名為Pgacs1-1。‘JJB’為Pgacs1-1/1-1同質結合體。系列PgACS1啟動子缺失分析顯示,ERE距離TSS ≧ 1 kb則無法進行轉錄作用調控。相反的,‘梨仔拔’(‘LTB’) 與其他更年型品種之PgACS1則無PgRE1插入,在核心啟動子區具有ERE順式調控元件,果實進入後熟期可大量表現,以產生大量ACC與乙烯(20℃合成率20.83 µL C2H4 kg−1 h−1),此等位基因(allele)稱為PgACS1。‘LTB’為PgACS1/1同質結合體。由‘珍珠拔’芽條變異而來的‘香蜜拔’(‘SMB’)卻是更年型的後熟特性,分析其PgACS1啟動子核苷酸序列顯示為一異質結合體,具有一個Pgacs1-1等位基因與一個僅有嵌入717 bp 5’端LTR區的等位基因(命名為Pgacs1-2),所以果實後熟期PgACS1基因表現可部分恢復,已可產生足量的ACC與乙烯(20℃生合成率1.76 µL C2H4 kg−1 h−1)促使果實順利後熟,故呈現更年型表現型,其後熟速度與貯運性介乎‘JJB’與‘LTB’之間。推測芽條變異導致Pgacs1-1嵌入的PgRE1發生基因重組移除編碼區與3’端LTR而產生Pgacs1-2,因其ERE順式元件與TSS距離短於1 kb,故能部分恢復基因表現。依據PgACS1啟動子序列設計專一性引子,並且成功的區別十種番石榴品種的PgACS1的基因型。這些引子可作為分子標記,用於幫助在進行番石榴育種計畫時,在苗期預測將來番石榴果實的後熟特徵。此外,本研究結果也揭示,由‘泰國拔’所衍生的非更年型品種,包含‘珍珠拔’,其果實非更年型後熟性狀皆因PgACS1啟動子遭移位轉位子PgRE1嵌入所致。 Guava (Psidium guajava L.) is an interesting plant material for fruit ripening research, because this species has cultivars with different ripening behaviors that associated with its postharvest storage life. The discrepancy in postharvest phenotypes is due to the distinct ability of system-2 ethylene biosynthesis; however, its molecular mechanism has not been studied in detail. PgACS1 (JQ280301), a key system-2 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) gene, is responsible for massive ethylene production during ripening in guava. This study investigated the relationship among the genotypes of PgACS1, ethylene production, and postharvest storage life of different guava cultivars. The PgACS1 promoter of nonclimacteric ’Jen-Ju Bar’ (’JJB’) guava was found to be interrupted by a 5,487 bp Ty1/Copia long-terminal repeat (LTR) retrotransposon, Psidium guajava retrotransposon 1 (PgRE1) (LC699797) at position -6. Moreover, PgRE1 contains conserved genes for gag, pol, integrase (INT), reverse transcriptase (RT), and RNase H (RH). The insertion was in the antisense transcription direction of PgACS1, resulting in the displacement of the Ethylene Response Element (ERE) cis-regulatory element from -146 ~ -139 to -5599 ~ -5606, upstream from the putative transcription start site (TSS). Therefore, the gene cannot be activated to generate sufficient ACC to support ethylene production (< 0.1 µL C2H4 kg−1 h−1 at 20 ℃) in green-mature ‘JJB’ fruits. As a consequence, ‘JJB’ fruit behaved in a nonclimacteric phenotype with a longer postharvest storability. Additionally, ‘JJB’ PgACS1 promoter is insensitive, irresponsive, and independent of exogenous propylene treatment, and loses the ethylene autocatalytic ability of the system-2 ACS gene. This allele is named Pgacs1-1 and‘JJB’possesses homozygous Pgacs1-1/1-1 genotype. A serial deletion analysis of PgRE1 in the ‘JJB’ Pgacs1-1 promoter displayed that ERE could not modulate the transcription if it is located more than or equal to 1 kb from the putative TSS. In contrast, the PgACS1 promoter of ’Li-Tzy Bar’ (’LTB’) and other climacteric guava cultivars that do not have a PgRE1 insertion but have an ERE cis-regulatory elements in their core promoter region. This allele is called PgACS1 and ‘LTB’is homozygous of PgACS1/1. It was highly expressed to promote massive ethylene emission (20.83 µL C2H4 kg−1 h−1 at 20 ℃) during the climacteric stage of ‘LTB’ fruit. ‘Shiang Mi Bar’ (‘SMB’), a bud sport mutant of ‘JJB’, surprisingly exhibits a climacteric manner of ripening. Analysis of the nucleotide sequence of its PgACS1 promoter showed that ‘SMB’ is heterozygous of Pgacs1-1and Pgacs1-2 (consisting of a 717 bp of 5'-LTR). The expression of ‘SMB’ PgACS1 was partially restored, rendering sufficient amounts of ACC and ethylene evolution (1.76 µL C2H4 kg−1 h−1 at 20 ℃) to enhance fruit ripening. Therefore, ‘SMB’ exhibited climacteric characteristics and its ripening speed and storability were between ’JJB’ and ’LTB’. It was hypothesized that genetic recombination of PgRE1 in the Pgacs1-1 removed the coding region and the 3'- LTR of PgRE1 resulting in a bud sport mutant, Pgacs1-2. Since the distance between the ERE cis-element and the putative TSS is shorter than 1 kb, the transcription level of ‘SMB’ PgACS1 was moderately recovered. Specific PCR primers targeting the PgACS1 promoter were designed and successfully determined the PgACS1 genotypes among ten guava cultivars that highly associated with their ethylene production. The primers were suggested for application as molecular markers to assist in early ripening-behavior screening among progenies of a guava breeding project. Moreover, this study has revealed that nonclimacteric character of guava cultivars from the same ancestor with ‘JJB’ are caused by retrotransposon insertion in the PgACS1 promoter. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/83221 |
DOI: | 10.6342/NTU202204034 |
全文授權: | 同意授權(全球公開) |
電子全文公開日期: | 2027-09-26 |
顯示於系所單位: | 園藝暨景觀學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-110-2.pdf 此日期後於網路公開 2027-09-26 | 3.96 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。