水稻台農67號及其香米突變體SA0420香味相關基因之探討

Study on aroma related genes in rice（Oryza sativa L.）Tainung 67 and its aromatic mutant, SA0420.

郭淑明(Shu-Ming Kuo)

指導教授 ： 闕甫伈 ； 王強生

亞洲大學/健康學院/生物科技學系碩士班/碩士(2005年)

摘要

稻米是全球主要糧食作物，本論文探討影響稻米品質的香味性狀及基因表現，進而選殖香味相關基因，期能提高水稻品質與商品價值。試驗採用水稻品種台農67 號 (TNG67) 及其疊氮化鈉誘變之香米突變品系SA0420 為材料，其葉片與穀粒於生育時期皆會產生香味。SA0420 的香味性狀，是由單一顯性基因座所控制，有別於大多數香米栽培種的隱性遺傳。初步實驗結果證實，澳洲團隊所發表的香味基因betaine aldehyde dehydrogenase 2 (BAD2) (Bradbury et al., 2005) 並非SA0420 突變體呈香的關鍵基因，且發現BAD2 亦非TNG71 (益全香米)、TNG67 突變庫中的香米突變體SA0418 與SA1613 呈香的基因。本論文以蛋白質體學策略獲得的差異性蛋白質中，選取與香味成分2-acetyl-1-pyrroline (2-AP) 生合成有關的差異性蛋白質glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 與glutamate-1-semialdehyde 2,1-aminotransferase (GSA-AT) 二個候選基因，進行序列比對分析，設計專一性引子，先以逆轉錄聚合酶連鎖反應（RT-PCR），探討基因之表現，再進行基因之選殖、定序。試驗結果發現：GAPDH 基因在水稻基因組中共有7套並加以命名，其中GAPDH7 與GSA-AT相當接近第八對染色體上與香味基因連鎖的SSR 標誌RG1。RT-PCR 結果發現，水稻GAPDH2 (XM_493811)與GSA-AT (XM_507903) 基因之表現在TNG67 與SA0420 二品系之差異並不明顯，可能因為香味成分閾值很低之故，推測香味性狀並非是質的差異，即基因的有或無，而是量的差異，即基因表現之強或弱所致；然而，水稻GAPDH 2 與GSA-AT 基因在穀粒中的基因表現無或極弱，為香味化合物在葉片中合成，而不在穀粒中合成的推論，提供了間接證據。選殖與定序GAPDH1 與GAPDH2 基因發現，SA0420 有數個核苷酸的突變，造成GAPDH1 與GAPDH2 胺基酸的改變，可能導致GAPDH 蛋白質活性較低或不表現，當SA0420 GAPDH 蛋白質的表現量減少時，glyceraldehyde-3-phosphate (GA3P) 會因GAPDH的作用減緩而累積，於是會增加由triose-phosphate isomerase (TPI) 的催化而形成dihydroxyacetone phosphate (DHAP) 的量，較多量的DHAP能與Δ1-pyrroline-5-carboxylate (P5C) 結合而形成2-AP，而使SA0420 呈香。由GSA-AT之選殖、定序及親緣分析之結果發現，能明顯將17 個香米及7 個非香米歸群，證明GSA-AT 是與香米香味有關的基因。

關鍵字

香米 ； 香米突變體SA0420 ； 疊氮化鈉誘變 ； 甜菜鹼乙醛去氫酶 ； 甘油醛-3-磷酸脫氫酶

並列摘要

Rice is an important staple food in the world aroma is one of its important traits providing higher value. The purpose of this thesis is to study the aromatic phenotype and to clone the related genes in order to improve rice eating quality. Rice cultivar Tainung 67 (TNG67) and its sodium azide induced mutants were used for experiments. A mutant, SA0420, produces aroma in leaves and grains at various stages of development and is conditioned by a single dominant locus that differs from most of the aroma rice varieties which were recessive traits. Our sequencing results showed that the aroma of SA0420 is not controlled by the betaine aldehyde dehydrogenase (BAD2) gene as reported by Bradbury et al. (2005). Besides, the aroma of variety TNG71 and the aromatic mutants, SA0418 and SA1613 of TNG67 mutation pool are not controlled by the BAD2 gene, either. Our proteomic studies discovered that two differentially expressed proteins glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glutamate-1-semialdehyde-2,1-amino- transferase (GSA-AT) may closely related to the aroma production (Chou, 2004). Sequences of rice GAPDH and GSA-AT genes were firstly obtained by bioinformatic approach, cloned and sequenced by PCR technology. A total of 7 GAPDH genes are found in the rice genome and named as GAPDH1 to GAPDH7 for the first time in this thesis. The GAPDH7 and GSA-AT genes are located very close to the RG1 SSR marker on the chromosome 8, which was previously reported to be linked to the aroma trait. Because the aroma produced in rice is in a quantitative but not a qualitative manner no significant difference was detected in the expression of GAPDH2 (XM_493811) and GSA-AT (XM_507903) in leaves of TNG67 and SA0420 by RT-PCR. Both GAPDH2 and GSA-AT genes are expressed at very low to none levels in rice grains as compared with those of leaves, providing an evidence for our previous hypothesis that the aroma of SA0420 mutant is mainly produced in leaves then translocate to grains during grain filling period (C. S. Wang unpublished data). Sequences analyses of GAPDH1 and GAPDH2 showed that mutations occurred in the GapA domain of GAPDH1, GAPDH2 may affect the structure and activity of proteins. The lack of two GAPDH proteins in SA0420 mutant could be a key factor to increase its aroma synthesis. The clear grouping by phylogenetic analyses of the GSA-AT sequences from 17 fragrant and 7 non-fragrant rice varieties also provides the evidence that the GSA-AT is related to the aroma of SA0420.

並列關鍵字

aromatic rice ； aromatic mutant, SA0420 ； sodium azide (NaN3) mutagenesis ； betaine aldehyde dehydrogenase, BAD ； glyceraldehyde-3-phosphate dehydrogenase, GAPDH ； glutamate-1-semialdehyde 2,1-aminotransferase, GSA-AT

參考文獻

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