番石榴果實ACC合成酶與八氫茄紅素合成酶基因的選殖與表現分析

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論文名稱: 番石榴果實ACC合成酶與八氫茄紅素合成酶基因的選殖與表現分析
研究生姓名: 姚鐙凱
指導教授姓名: 陳幼光
出版年: 2009
學校名稱: 國立屏東科技大學
系所名稱: 農園生產系所
關鍵字: 番石榴;ACC合成酶;八氫茄紅素合成酶;分子標記;guava;ACC synthase;phytoene synthase;molecular marker
摘要: 番石榴(Psidium guajava L.)依果實後熟期間呼吸速率與乙烯生成量,可分為更年性與非更年性果實二類。為瞭解此二類果實於分子表達層次上的差異,本實驗分別從二類果實中選殖出乙烯生合成途徑中的ACC合成酶(ACC synthase, ACS)基因序列進行比較分析。不論更年性或非更年性的番石榴ACS cDNA序列皆具1,476 bp 開放解讀區域,可推譯出491個胺基酸,經結構分析發現其C端具有胺基轉移酶(Aminotransferase)區域、鈣離子依賴型蛋白激酶(Calcium-Dependent Protein Kinase, CDPK)作用之位置與有絲分裂原活化蛋白激酶(Mitogen-Activated Protein Kinase 6, MAPK6)的磷酸化部位三個區域。另番石榴果肉顏色也有紅肉與白肉的差別,而紅肉番石榴的色素主要屬於類胡蘿蔔素類,為找出區別二種番石榴的原因,本實驗也針對類胡蘿蔔素生合成途徑上的八氫茄紅素合成酶(Phytoene synthase,PSY)基因進行選殖,並從紅肉番石榴中選殖出PgPSY1-1,其開放解讀區具有804bp,可推譯出267個胺基酸;而白肉番石榴所選殖到的PgPSY1-2其開放解讀區則具有654 bp,可推譯出217個胺基酸。番石榴PgPSY1-1與PgPSY1-2胺基酸序列經比對確認為異戊二烯焦磷酸合成酶(Trans-isoprenyl diphosphate synthases)家族的一員,經分析發現PgPSY1-1的序列中具有基質結合區(substrate binding pocket)、鎂離子結合部位(substrate-Mg2+ binding site)、催化殘基(catalytic residues)、富含天門冬胺酸區域(aspartate-rich region)及活化位置蓋子殘基(active site lid residues)等的保守區域,而在PgPSY1-2的序列則缺乏活化位置殘基區域。Fruits of guava (Psidium guajava L.) have been categorized into climacteric and non-climacteric based on respiratory rate and ethylene production during ripening. To explore the molecular differences of gene expression between climacteric and non-climacteric fruits, both gene sequences of an ACC synthase (ACS) from the two types of fruits were cloned and compared. The full length ORF of the cloned ACS cDNA was 1,476bp encoding a polypeptide of 491 amino acids. The C-terminal amino acid sequence of PgACS1 contains an aminotransferase region, a calcium-dependent protein kinase (CDPK) region, and a mitogen-activated protein kinase 6 (MAPK6) region. Flesh color characteristics of guava fruit is mostly classified as red and white. Red pulp’s pigment is primarily derived from carotenoids. To distinguish between the two types of flesh color, phytoene synthase (PSY) gene sequences were cloned and analyzed. The full length ORF of the cloned PgPSY1-1 cDNA was 804 bp encoding a polypeptide of 267 amino acids from red fleshed guava. On the other hand, the full length ORF of the cloned PgPSY1-2 cDNA from white fleshed guava was 654 bp encoding a polypeptide of 217 amino acids. PgPSY1-1 and PgPSY1-2 amino acid sequences belong to trans-isoprenyl diphosphate synthases family. Homology search revealed that the predicted PgPSY1-1 amino acid sequence contains a substrate binding pocket, a substrate-Mg2+ binding site, catalytic residues, an aspartate-rich region, and an active site lid residue domain. The PgPSY1-2 amino acid sequence lacks the active site lid residue domain.
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