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標題: 利用蛋白質體技術探討水稻台農67號及其香米突變體SA0420之差異性蛋白質
Characterization of rice(Oryza sativa L.) Tainung 67 and its aromatic mutant SA0420 using proteomic approach
作者: 周思儀
Chou, Si-Yi
關鍵字: proteomics;蛋白質體學;aromatic rice;香米
出版社: 農藝學系
稻米是全球主要糧食作物,而香味為稻米重要品質性狀之一,故香米在全世界許多地區均有很高的商品價值。本試驗採用水稻品種台農67號(TNG67)及其疊氮化鈉(sodium azide, NaN3)誘變之香米突變品系SA0420為材料,SA0420為單一顯性基因座的香味遺傳突變體,其葉片與穀粒於生育時期皆會產生香味,且香味會因時期不同而有所變化,因此本研究以蛋白質體學的方法,於蛋白質表現層次探討TNG67與香米突變品系SA0420在不同生育時期之葉片與穀粒間的蛋白質表現差異,期能發現與香味合成相關之蛋白質。試驗結果發現,可能與香味合成相關之蛋白質包括glyceraldehyde-3-phosphate dehydrogenase(GAPDH)(L3、L4、L5)及glutamate-1-semialdehyde 2,1-aminomutase(GSA-AT)(L8);可能與葉尖黃化相關之蛋白質包括rubisco次單位結合蛋白質(rubisco subunit binding-protein alpha subunit)(L1)與蛋白質雙硫異構酶(protein disulfide isomerase, PDI)(L2)等;可能與逆境相關之蛋白質則包括草酸氧化酶(oxalate oxidase)(L10)、14.3.3蛋白質(L11)、與葡萄糖基轉移酶-10相似的蛋白質(glucosyltransferase-10-like protein)(G2)、短鏈的酒精脫氫酶(short-chain alcohol dehydrogenase, ADH)(G4)、LEA蛋白質(late embryogenesis abundant proteins)(G6)、富含脯胺酸之蛋白質同源物WCOR518(proline rich protein homolog, WCOR518)(G7)、水分逆境下誘導的蛋白質LP3-2(water-stress-inducible protein LP3-2)(G8)、真菌誘發的蛋白質CMPG1(fungal elicitor protein, CMPG1)(G9)以及富含甘胺酸的RNA結合蛋白質2(glycine-rich RNA-binding protein 2)(G10)等;獨特存在於TNG67穀粒中的starch granule-bound starch synthase(GBSS)(G1)為與澱粉合成相關之蛋白質,其他尚有未知的蛋白質L6,質體核糖體蛋白質2之前驅物(plastid-specific ribosomal protein 2 precursor)(L7)、cyclin A-type(L9)、絲胺酸O-乙醯轉移酶(serine O-acetyltransferase)(G3)以及未知蛋白質(G5)等。其中GAPDH為催化fructose-1,6-bisphosphate形成glyceraldehyde 3-phosphate(GA3P)的重要酵素,而GA3P與合成香味重要成分2-acetyl-1-pyrroline(2-AP)的重要前驅物dihydroxyacetone phosphate(DHAP)互為isoform,因此GAPDH可能為與香味合成相關之蛋白質。此外,L-glutamate形成glutamate-1-semialdehyde後,會經GSA-AT催化形成5-aminolevulinate(ALA),但由於合成香味成分2-AP的重要中間產物Δ1-pyrroline-5-carboxylic acid(P5C)係由L-glutamate形成L-glutamyl-γ-phosphate後而形成,因此推測GSA-AT雖未直接參與P5C合成,但其可能間接提供合成P5C途徑之受質。因此未來研究將選殖出相對應的全長基因,進行基因表現與功能分析,探討其與突變香味性狀之相關性,以確定香味合成的作用機制,再利用基因轉殖方法,期能直接確認目標基因與外表型的關聯。

Rice is an important staple food in world. Aroma is one of the important quality trait and results in a high price in the market. In this study, the rice cultivar Tainung 67 (TNG67) and its aromatic mutant SA0420 derived from sodium azide (NaN3) mutagenesis were used for experiment. In SA0420, the aroma is controlled by a single dominant locus and generates aroma at various developmental stages in leaves and grains. Proteomics is powerful to investigate the relationship between protein profiles and the phenotype, and was applied to study the differential protein(s) and their relationships with aroma in the leaves and grains of TNG67 and SA0420.
Several differentially expressed proteins were separated and identified by MS/MS and blast analysis. Firstly, proteins with clues related to aroma producton include spot L3, L4 and L5, matched with glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) and spot L8, matched with glutamate-1-semialdehyde 2,1-aminomutase (GSA-AT), respectively. Secondly, the proteins which may related to SA0420's yellow leaf tip color are matched with rubisco subunit binding-protein alpha subunit (L1) or protein disulfide isomerase (PDI) (L2). In addition, some identified proteins such as oxalate oxidase (L10), 14.3.3 protein (L11), glucosyltransferase-10-like protein (G2), short-chain alcohol dehydrogenase (ADH) (G4), late embryogenesis abundant (LEA) proteins (G6), proline rich protein homolog WCOR518 (G7), water-stress-inducible protein LP3-2 (G8), fungal elicitor protein CMPG1 (G9), and glycine-rich RNA-binding protein 2 (G10), etc., may response to stresses. Moreover, protein related to starch biosynthesis, starch granule-bound starch synthase (GBSS) (G1), was identified only in the grains of TNG67. Other proteins including plastid-specific ribosomal protein 2 precursor (L7), cyclin A-type (L9), serine O-acetyltransferase (G3), and two still unknown proteins (L6 and L5) were also identified.
The major aroma compound, 2-acetyl-1-pyrroline (2-AP), in rice is proposed to be synthesized from dihydroxyacetone phosphate (DHAP) or glyceraldehyde 3-phosphate (GA3P) converting from fructose- 1,6-bisphosphate. Therefore, the lack of GAPDH may be a key factor leads to the aroma synthesis in the SA0420 mutant. Because the compound, Δ1-pyrroline-5-carboxylic acid (P5C), from L-glutamate is also the key intermediate of 2-AP synthesis pathway. Glutamate- 1-semialdehyde synthesized from L-glutamate can be converted to 5-aminolevulinate (ALA) and further catalyzed by GSA-AT. Therefore, GSA-AT is speculated to involve in the synthesis of P5C providing a substrate for 2-AP production. Further studies will be conducted to clone related genes from mutant and to investigate their functions and relationships with aroma production by transgenic plant approach.
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