不同後熟特性番石榴ACC合成酶啟動子之選殖、分析與應用

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論文名稱: 不同後熟特性番石榴ACC合成酶啟動子之選殖、分析與應用
研究生姓名: 趙晨崴
指導教授姓名: 吳俊達
出版年: 2014
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;後熟;反轉錄轉位子;啟動子;guava;acc synthase;ripening;retrotransposon;promoter
摘要: ‘珍珠拔’番石榴(Psidium guajava L.)呈現非更年型後熟特性源於‘系統二’ACC合成酶基因PgACS1無法表現,而缺乏大量自動催化乙烯合成。利用染色體步移(Genomic Walking)技術,分別選殖‘珍珠拔’與更年型‘梨仔拔’PgACS1、2基因組選殖系進行比對分析。兩參試品種PgACS1轉錄區域(transcribed region)皆為3120 bp,其中包含三個插入子(intron)呈現典型ACS基因結構,其核苷酸序列相同度為99%。反之,‘梨仔拔’PgACS1(LTB PgACS1)與‘珍珠拔’(JJB PgACS1)啟動子(promoter)核苷酸序列相同度僅為48%,將兩段序列於NCBI資料庫進行比對,LTB PgACS1啟動子並無相似序列;JJB PgACS1啟動子則可比對到多種物種相似序列,且皆集中在-2315~-800區域,相同度皆介於64~71%間,其中比對得分最高之序列為葡萄反轉錄轉位子(retrotransposon)V12,此結果暗示JJB PgACS1啟動子序列異於LTB PgACS1者可能源於反轉錄轉位子插入所致。以PLACE軟體分析啟動子,LTB PgACS1具有2個 ERE(ethylene response element)順式元件(cis-element);反之, JJB PgACS1選殖出的2310 bp啟動子區域則無任何ERE存在。生長調節劑indole-3-acetic acid、gibberellinA4+7、6-benzylaminopurine、乙烯、abscisic acid、methyl jasmonate處理,皆可誘導‘梨仔拔’綠熟果PgACS1表現,然而‘珍珠拔’綠熟果PgACS1則表現低,推測因反轉錄轉位子插入JJB PgACS1啟動子造成調控關鍵區域順式元件大規模位移所致。以PgACS1驅動β-glucuronidase(GUS)報導基因暫時性表現評估啟動子活性,JJB PgACS1啟動子在‘梨仔拔’與‘珍珠拔’果實背景活性差異不顯著,且LTB PgACS1啟動子活性於兩參試品種果實背景皆高於JJB PgACS1啟動子,暗示PgACS1品種間表現量差異主要源於啟動子順式元件變異而非反式元件(trans-element)因素所致。依據PgACS1序列設計專一性PCR引子,以葉片抽取DNA為模板,可成功鑑別10個番石榴品種後熟特性,可應用於育種後裔果實後熟特性早期篩選分子標誌。‘Jen-Ju Bar’ (JJB) guava(Psidium guajava L.)exhibited a nonclimacteric ripening behavior resulting from lacking ofPgACS1, a System Ⅱ ACC synthase (ACS) gene, expression and, therefore, autocatalytic ethylene production. Genomic clones of PgACS1 isolated from ‘Jen-Ju Bar’ and ‘Li-Tzy Bar’ (LTB) guava, a climacteric variety, via Genomic Walking technique were alignmented and analyzed. The transcribed regions for both clones were 3120 bp cotaining 3 introns, which displayed a typical ACS characteristic, and sharing 99% nucleotide sequence identity. However, the identity of promoter sequences of LTB PgACS1 and JJB PgACS1 was only 46%. Blasting analysis on NCBI database showed no similar sequence for the promoter of LTB PgACS1. In contrast, several hits from various species similar to the -2310 ~ -800 promoter region of JJB PgACS1 with 64~71% identity were found; and grape (Vitis vinifera L.) retrotransposon V12 possessed the highest score, which implied that the sequence differences of PgACS1promoter region between LTB and JJB were very likely caused by insertion of a retrotransposon. The promoter sequences of LTB and JJB PgACS1 analyzed by PLACE software revealed that there were two Ethylene Response Elements (ERE) in the former; and no ERE was detected in the isolated promoter region (-1~ -2310) of the latter. PgACS1was up-regulated in mature-green LTB fruit after all treatments of plant growth regulators tested, including indole-3-acetic acid, gibberellin A4+7, 6-benzylaminopurine, ethylene, abscisic acid, and methyl jasmonate; but no or low response of the gene expression in the case of JJB fruit, which may be a result from massive shift of the cis-elements after retrotransposon insertion. The facts that the activity of JJB PgACS1 promoter displayed no significant difference between LTB and JJB fruit background in transient expression assay ofβ-glucuronidase (GUS) reporter gene driven byPgACS1 promoters, as well as that LTB PgACS1promoter was more active than that of JJB one in both fruit backgrounds, suggested the cis-element difference, rather than trans-element variation, was the major reason of the activity variation of PgACS1promoterbetween the cultivars tested. Based on the results of PCR assessments, which utilized specific PCR primers designed according to PgACS1 genomic clones and the DNA extracted from guava leaves, successfully discriminated the ripening behavior among the 10 guava cultivars examined, the primers was suggested to apply as molecular markers to assist early ripening-behavior screening among progenies of a guava breeding project.
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